Sensors, systems and methods for detecting analytes using same

ABSTRACT

Sensors, as well as systems and methods of using the same are provided. Aspects of the sensors include a piezoelectric base, a plurality of surface-associated compositions that are stably associated with the piezoelectric base, and a plurality of crosslinking compositions that are configured to crosslink one or more surface-associated compositions in the presence of an analyte. The sensors, systems and methods described herein find use in a variety of applications, including the detection of an analyte in a sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of the filing date of U.S.Provisional Patent Application Ser. No. 62/086,036, filed on Dec. 1,2014, the disclosure of which application is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention concerns sensors, systems and methods that finduse in the detection of analytes.

BACKGROUND

Fast and accurate detection and quantification of target analytes, suchas biological analytes, continues to be a major challenge in the fieldof bioanalytical chemistry. Frequently, analytical techniques requiresignificant amounts of time for sample preparation and analysis, leadingto significant delays between sample acquisition and reporting of testresults. For example, current techniques for detecting micro-organismsin a sample frequently require culturing a sample for extended periodsof time, often several days, in order to confirm the presence or absenceof the micro-organism. Thus, there remains a need for sensors, systemsand methods that can facilitate rapid and accurate analysis of targetanalytes.

SUMMARY

Sensors, as well as systems and methods of using the same are provided.Aspects of the sensors include a piezoelectric base, a plurality ofsurface-associated compositions that are stably associated with thepiezoelectric base, and a plurality of crosslinking compositions thatare configured to crosslink one or more surface-associated compositionsin the presence of an analyte. The sensors, systems and methodsdescribed herein find use in a variety of applications, including thedetection of an analyte in a sample.

Aspects of the invention include sensors that include a piezoelectricbase, a plurality of surface-associated compositions that are stablyassociated with the piezoelectric base, and a plurality of crosslinkingcompositions that are configured to crosslink one or moresurface-associated compositions in the presence of an analyte.

In some embodiments, the piezoelectric base includes at least oneelectrode. In some embodiments, a sensor includes an oscillator circuitthat is electrically connected to the at least one electrode, and isconfigured to drive the sensor at one or more frequencies. In someembodiments, the oscillator circuit includes an automatic gain control(AGC) portion. In some embodiments, the surface-associated compositionsinclude one or more of: protein A, protein G, protein A/G, or protein L.In some embodiments, the surface-associated compositions include one ormore polyclonal antibodies. In some embodiments, the crosslinkingcompositions include one or more of: protein A, protein G, protein A/G,or protein L. In some embodiments, the crosslinking compositions includeone or more polyclonal antibodies. In some embodiments, a sensorincludes a computer-readable medium that contains a plurality of storeddata. In some embodiments, the stored data includes a calibration valuefor the sensor. In some embodiments, the stored data includes an analytesignature. In some embodiments, the stored data includes an operatingparameter for the sensor. In some embodiments, the piezoelectric baseincludes a quartz crystal. In some embodiments, the quartz crystal is anAT-cut quartz crystal. In some embodiments, the piezoelectric base has asurface texture. In some embodiments, the at least one electrode has aninterdigitated structure. In some embodiments, a plurality of thesurface-associated compositions and/or crosslinking compositionsincludes a detectable label.

Aspects of the invention include systems for detecting the presence ofan analyte in a sample, the system including a sensor that includes apiezoelectric base, a plurality of surface-associated compositions thatare stably associated with the piezoelectric base, a plurality ofcrosslinking compositions that are configured to crosslink one or moresurface-associated compositions in the presence of the analyte, and atleast one electrode, as well as an oscillator circuit that iselectrically connected to the at least one electrode and is configuredto drive the sensor at one or more frequencies, wherein the oscillatorcircuit includes an automatic gain control (AGC) portion, a detectionunit configured to receive a plurality of data from the oscillatorcircuit, and a processor configured to analyze the data received fromthe oscillator circuit and to detect the presence of the analyte in thesample.

In some embodiments, the sensor, the oscillator circuit, the detectionunit, and the processor are formed into a single device. In someembodiments, the system includes a graphical user interface. In someembodiments, the device is a hand-held device. In some embodiments, thesensor and the detection unit are separate elements, and the sensor isadapted to connect to the detection unit. In some embodiments, thesystem includes a plurality of sensors and a plurality of oscillatorcircuits that are configured to drive each of the sensors at one or morefrequencies. In some embodiments, the detection unit includes afrequency spectrum analyzer. In some embodiments, the detection unit isconfigured to transmit an analysis result obtained from the sample to aseparate location. In some embodiments, the detection unit is configuredto wirelessly transmit the analysis result to the separate location. Insome embodiments, the analysis result includes a geographical locationfrom which the sample was collected. In some embodiments, thesurface-associated compositions include one or more of: protein A,protein G, protein A/G, or protein L. In some embodiments, thesurface-associated compositions include one or more polyclonalantibodies. In some embodiments, the crosslinking compositions includeone or more of: protein A, protein G, protein A/G, or protein L. In someembodiments, the crosslinking compositions include one or morepolyclonal antibodies. In some embodiments, a system includes acomputer-readable medium that contains a plurality of stored data. Insome embodiments, the computer-readable medium is located in the sensor.In some embodiments, the computer-readable medium is located in thedetection unit. In some embodiments, the stored data includes acalibration value for the sensor. In some embodiments, the stored dataincludes an analyte signature. In some embodiments, the stored dataincludes an operating parameter for the sensor. In some embodiments, thepiezoelectric base of the sensor includes a quartz crystal. In someembodiments, the quartz crystal is an AT-cut quartz crystal. In someembodiments, the piezoelectric base of the sensor has a surface texture.In some embodiments, the at least one electrode has an interdigitatedstructure. In some embodiments, a plurality of the surface-associatedcompositions and/or crosslinking compositions includes a detectablelabel. In some embodiments, a system includes a detection device that isconfigured to detect the detectable label. In some embodiments, thedetection device is a spectrophotometer, a fluoroscope, or anellipsometer.

Aspects of the invention include methods for detecting the presence ofan analyte in a sample, the methods involving contacting a sensor withthe sample, wherein the sensor includes a piezoelectric base, aplurality of surface-associated compositions that are stably associatedwith the piezoelectric base, a plurality of crosslinking compositionsthat are configured to crosslink one or more surface-associatedcompositions in the presence of the analyte, and at least one electrode,applying a current to an oscillator circuit that is electricallyconnected to the at least one electrode and is configured to drive thesensor at one or more frequencies, wherein the oscillator circuitincludes an automatic gain control (AGC) portion, measuring one or moreparameters of the sensor and/or oscillator circuit as a function oftime, and analyzing the one or more parameters of the sensor and/oroscillator circuit to detect the presence of the analyte in the sample.

In some embodiments, the methods involve detecting whether the analyteis present in the sample at a concentration that is above a thresholdconcentration. In some embodiments, the methods involve contacting thesample with an activated carbon composition before contacting the samplewith the sensor. In some embodiments, the activated carbon compositionincludes a carbon nanotube. In some embodiments, the methods involvedriving the sensor at a plurality of frequencies, and measuring one ormore parameters of the sensor and/or oscillator circuit as a function oftime at each frequency. In some embodiments, measuring the one or moreparameters of the sensor involves measuring a frequency, an amplitude,and/or a frequency bandwidth of a waveform that is generated in thesensor. In some embodiments, measuring the one or more parameters of theoscillator circuit involves measuring a voltage, a resistance, anadmittance, an impedance, or a conductance value of the automatic gaincontrol (AGC) portion of the oscillator circuit. In some embodiments,analyzing the one or more parameters of the sensor and/or oscillatorcircuit involves comparing the one or more parameters to a calibrationvalue. In some embodiments, analyzing the one or more parameters of thesensor and/or oscillator circuit involves comparing the one or moreparameters to an analyte signature. In some embodiments, the one or moreparameters of the sensor and/or oscillator circuit are measured andanalyzed to detect the presence of the analyte in the sample in lessthan 10 seconds after the sensor is contacted with the sample. In someembodiments, a plurality of the surface-associated compositions and/orcrosslinking compositions includes a detectable label, and the methodinvolves detecting the detectable label using a detection device.

Aspects of the invention include methods of making a sensor, the methodsinvolving depositing a plurality of surface-associated compositions on apiezoelectric base, wherein the plurality of surface-associatedcompositions are adapted to stably associate with the piezoelectricbase, and depositing a plurality of crosslinking compositions on top ofthe surface-associated compositions, wherein the crosslinkingcompositions are configured to crosslink one or more of thesurface-associated compositions in the presence of an analyte.

In some embodiments, the surface-associated compositions include one ormore of: protein A, protein G, protein A/G, or protein L. In someembodiments, the surface-associated compositions include one or morepolyclonal antibodies. In some embodiments, the crosslinkingcompositions include one or more of: protein A, protein G, protein A/G,or protein L. In some embodiments, the crosslinking compositions includeone or more polyclonal antibodies. In some embodiments, depositing theplurality of surface-associated compositions on the piezoelectric baseinvolves contacting the piezoelectric base with a solution that includesthe plurality of surface-associated compositions. In some embodiments,the solution that includes the plurality of surface-associatedcompositions has a concentration of surface-associated compositionsranging from 0.1 μg/mL to 10 mg/mL. In some embodiments, depositing theplurality of crosslinking compositions on top of the surface-associatedcompositions involves contacting the plurality of surface-associatedcompositions with a solution that includes the plurality of crosslinkingcompositions. In some embodiments, the solution that includes theplurality of crosslinking compositions has a concentration ofcrosslinking molecules ranging from 0.1 μg/mL to 10 mg/mL. In someembodiments, the plurality of surface-associated compositions and/or theplurality of crosslinking compositions includes a detectable label.

Aspects of the invention include methods of making a sensor, the methodsinvolving depositing a plurality of first molecules on a surface of apiezoelectric base, wherein the plurality of first molecules areconfigured to stably associate with the piezoelectric base, and are alsoconfigured to stably associate with one or more second molecules,depositing a plurality of the second molecules on top of the pluralityof first molecules, and allowing the first and second molecules toself-assemble into a plurality of surface-associated compositions and aplurality of crosslinking compositions, wherein each of thesurface-associated compositions and the crosslinking compositions eachcontain at least one first molecule and an least one second molecule. Insome embodiments, first molecule is protein A, protein G, protein A/G,or protein L, and the second molecule is a polyclonal antibody.

Aspects of the invention include kits that include two or more sensorspackaged in a sterile package, wherein each sensor includes apiezoelectric base, a plurality of surface-associated compositions thatare stably associated with the piezoelectric base, and a plurality ofcrosslinking compositions that are configured to crosslink one or moresurface-associated compositions in the presence of an analyte.

In some embodiments, the first sensor is adapted to detect a firstanalyte, and the second sensor is adapted to detect a second analytethat is different from the first analyte. In some embodiments, aplurality of the surface-associated compositions and/or crosslinkingcompositions on the two or more sensors includes a detectable label, andthe kit includes a reagent that is configured to facilitate detection ofthe detectable label. In some embodiments, a kit includes an activatedcarbon composition. In some embodiments, the activated carboncomposition includes a carbon nanotube. In some embodiments, a kitincludes one or more sample collection devices.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing a schematic representation of a subjectsensor with a plurality of surface-associated compositions andcrosslinking compositions thereon.

FIG. 2 is a diagram showing a schematic representation of a subjectsensor with a plurality of surface-associated compositions andcrosslinking compositions thereon shortly after the sensor has beencontacted with a sample containing an analyte.

FIG. 3 is a diagram showing a schematic representation of a subjectsensor with a plurality of surface-associated compositions andcrosslinking compositions thereon after the sensor has been contactedwith a sample containing an analyte.

FIG. 4 is a diagram showing a schematic representation of a subjectsensor with a plurality of surface-associated compositions andcrosslinking compositions thereon after the sensor has been contactedwith a sample containing an analyte.

FIG. 5 is a diagram showing a schematic representation of a subjectsensor electrically connected to a system that includes electricalcircuitry for driving the sensor and detecting data from the sensor.

FIG. 6 is a diagram showing a schematic representation of a signalreceived from a subject sensor as a function of time after the sensorhas been contacted with a sample containing an analyte.

FIG. 7 is a schematic illustration of a sensor and a detectioncomponent.

FIG. 8 is an illustration of that shows the flow of information betweenvarious components of a system.

FIG. 9 is a graph showing delta frequency response of as a function ofconcentration of Salmonella Heidelberg.

FIG. 10 is a table showing Log CFUs/mL and delta frequency response(mean, standard deviation (SD) and coefficient of variation (CV %)) forthe data provided in FIG. 9.

FIG. 11 is a graph showing delta frequency response as a function oftime for two Salmonella Heidelberg test solutions having differentconcentrations.

FIG. 12 is a table showing time in minutes, and the corresponding deltafrequency mean and CV % values for test solutions having two differentconcentrations of Salmonella Heidelberg.

FIG. 13 is a table showing delta frequency response and CV % values fordifferent sensors and different test solutions.

FIG. 14 is a graph showing delta frequency response as a function oftime for sensors having three different combinations of components.

FIG. 15 is a table showing the delta frequency mean and CV % values forsolutions having the indicated combination of components.

FIG. 16 is a diagram showing a schematic representation of a sensor madewith biotin-protein G surface compositions and crosslinking components.

FIG. 17 is a table showing delta frequency mean and CV % values forsensors that were made using a 100 ug/mL biotin-protein G solution.

FIG. 18 is a table showing delta frequency mean and CV % values forsensors that were made using a 10 ug/mL biotin-protein G solution.

FIG. 19 is a table showing delta frequency mean and CV % values forliquid state and dry sensors tested with Salmonella Typhimuriam testsolutions, or PBS.

FIG. 20 is a table showing ELISA results for two different Salmonellaserovars.

FIG. 21 is a table showing delta frequency mean and CV % values forsensors and test solutions with and without activated carbon.

FIG. 22 is a flow diagram that shows the steps of a method fordetermining a concentration of a target analyte in an unknown sampleusing an Internet-enabled system.

FIG. 23 is a flow diagram that shows the steps of a method fordetermining a concentration of a target analyte in an unknown sampleusing a non-Internet-enabled system.

DETAILED DESCRIPTION

Sensors, as well as systems and methods of using the same are provided.Aspects of the sensors include a piezoelectric base, a plurality ofsurface-associated compositions that are stably associated with thepiezoelectric base, and a plurality of crosslinking compositions thatare configured to crosslink one or more surface-associated compositionsin the presence of an analyte. The sensors, systems and methodsdescribed herein find use in a variety of applications, including thedetection of an analyte in a sample.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

In further describing various aspects of embodiments of the invention ingreater detail, aspects of the systems and devices of variousembodiments are reviewed first in greater detail, followed by adiscussion of methods and kits according to certain embodiments of theinvention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

Sensors

Aspects of the invention include sensors that are configured to detectan analyte in a sample. The subject sensors include a piezoelectricbase, a plurality of surface-associated compositions that are stablyassociated with the piezoelectric base, and a plurality of crosslinkingcompositions that are configured to crosslink one or moresurface-associated compositions in the presence of an analyte. Each ofthese elements is described in detail below.

Surface-Associated Compositions

Surface-associated compositions in accordance with embodiments of theinvention are configured to stably associate with a piezoelectric basewhile maintaining the ability to specifically bind to an analyte. By“stable association” or “stably associate” is meant that a firstmolecule or a portion thereof (e.g., a moiety) is bound to or otherwiseassociated with a second molecule, or with a structure (e.g., a surfaceof a substrate) under standard conditions. In certain instances, astable association may create one or more bonds between the first andsecond molecules, or between the first molecule and the structure, whichbonds may include, e.g., covalent or non-covalent interactions, such as,but not limited to, ionic bonds, hydrophobic interactions, hydrophilicinteractions, hydrogen bonds, van der Waals forces, (e.g., Londondispersion forces), dipole-dipole interactions, and the like. In someembodiments, the affinity between a first and a second molecule, orbetween a first molecule and a structure, is characterized by a K_(D)(dissociation constant) ranging from 10⁻⁴ M to 10⁻¹⁵ M, such as 10⁻⁵,10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³, or 10 ⁻¹⁴ M. Theterm “affinity” as used herein refers to the strength of the interactionbetween a first and a second molecule, or between a first molecule and asurface, wherein increased binding affinity is characterized by a lowerK_(D) value.

In some embodiments, a surface-associated composition may be composed ofa single molecule that is configured to stably associate with thepiezoelectric base and to specifically bind to an analyte. In someembodiments, a surface-associated composition may include a plurality ofdifferent molecules, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 or moremolecules. In such embodiments, the molecules that make up thesurface-associated composition are configured to associate with oneanother to form the composition. For example, in some embodiments, asurface-associated composition may include a first molecule that isconfigured to stably associate with a piezoelectric base, and a secondmolecule that is configured to specifically bind to an analyte. In suchembodiments, the first molecule is configured to stably associate withthe piezoelectric base, and is also configured to stably associate withthe second molecule to form a surface-associated composition.

Surface-associated compositions in accordance with embodiments of theinvention include one or more surface binding domains that areconfigured to stably associate with a substrate (e.g., a piezoelectricbase). In some embodiments, a surface binding domain may include: acyanogen bromide linkage (e.g., a cyanate ester linkage); an NHS esterlinkage; an aldehyde linkage; an azlactone ring linkage; a carbonyldiimidazole linkage; a sulfhydryl (thiol) linkage; a maleimide linkage;an iodoacetyl linkage; a pyridyl disulfide linkage; a hydrazide linkage;or a carbodiimide linkage.

In some embodiments, a surface binding domain is configured to adsorb toa surface (e.g., a surface of a piezoelectric base, or a surface of anelectrode) to stably associate with the surface. In some embodiments,the material properties of the surface may be configured to promotestable association with the surface binding domain. For example, in someembodiments, the surface material, surface energy, texture, and/orcharge distribution on the surface may be selected and/or modulated topromote stable association with a surface binding domain.

In some embodiments, a surface-associated composition may include one ormore polypeptides that have a surface binding domain. In suchembodiments, the surface binding domain of the polypeptide is configuredto stably associate with the piezoelectric base of the sensor. Examplesof polypeptides that include a surface binding domain include, but arenot limited to: protein A, protein G, protein A/G and protein L.

In some embodiments, a surface-associated composition includes two ormore molecules that are configured to stably associate with one anotherto form the surface-associated composition. In such embodiments, atleast one of the molecules includes at least one binding domain that isconfigured to form a stable association between the two or moremolecules. For example, in some embodiments, a surface-associatedcomposition may include a first molecule that includes a surface bindingdomain configured to form a stable association with the piezoelectricbase, and also includes a binding domain that is configured to stablyassociate with at least one other molecule (e.g., an analyte bindingmolecule) to form a surface-associated composition.

In some embodiments, a surface-associated composition may include afirst molecule that includes a plurality of surface binding domains thatare configured to form a stable association with the piezoelectric base,and also includes a plurality of binding domains that are configured tostably associate with a plurality of other molecules to form asurface-associated composition. For example, in some embodiments, asurface-associated composition may include a first molecule that has 2,3, 4, 5, 6, 7, 8, 9 or 10 or more surface binding domains that areconfigured to form a stable association with the piezoelectric base, andalso has 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more binding domains that areeach configured to stably associate with another molecule (e.g., ananalyte-binding molecule) to a form a surface-associated composition.

In some embodiments, a surface-associated composition includes a proteinmolecule that has a surface binding domain and also includes one or moreimmunoglobulin binding domains that are configured to form a stableassociation with an immunoglobulin molecule (e.g., an antibody).Examples of protein molecules that include at least one immunoglobulinbinding domain include, but are not limited to: protein A, protein G,protein A/G and protein L.

Surface-associated compositions in accordance with embodiments of theinvention include one or more analyte binding domains that areconfigured to specifically bind to an analyte. Specific binding betweenan analyte and an analyte binding domain results in the formation of astable association between the analyte and the analyte binding domain.Depending on the nature of the analyte, an analyte binding domain mayinclude, for example, a member of a receptor/ligand pair; aligand-binding portion of a receptor; a member of an antibody/antigenpair; an antigen-binding fragment of an antibody; a hapten; a member ofa lectin/carbohydrate pair; a member of an enzyme-substrate pair;biotin/avidin; biotin/streptavidin; digoxin/antidigoxin; a member of aDNA or RNA aptamer binding pair; a member of a peptide aptamer bindingpair; a member of a metal/metal-binding peptide pair; a chelating agent;and the like.

In some embodiments, an analyte binding domain of a surface-associatedcomposition includes an antigen. The antigen may specifically bind to ananalyte in a sample, such as an antibody of interest in the sample, or afragment thereof. In some embodiments, an analyte binding domainincludes an antibody, or an antibody fragment. The antibody mayspecifically bind to an analyte of interest in a sample, such as anantigen of interest in the sample.

In some embodiments, a sensor includes a plurality of surface-associatedcompositions whose analyte binding domains recognize different bindingregions of an analyte. As such, in certain embodiments, two or moredifferent analyte binding domains are each configured to bind todifferent portions of the same analyte (e.g., different epitopes on thesame antigen).

In some embodiments, an analyte binding domain of a surface-associatedcomposition includes an antibody. Antibodies in accordance withembodiments of the invention may be monoclonal or polyclonal, and may beany suitable class (isotype), including IgA, IgD, IgE, IgG or IgM.Antibodies in accordance with embodiments of the invention may also beany suitable subclass, including but not limited to IgG1, IgG2, IgG3 orIgG4. Antibodies may be produced by any suitable means, including butnot limited to, inoculation of a suitable mammal with an antigen,followed by recovery and purification of the antibody, or through theuse of recombinant antibody production technology. Antibodies may alsobe obtained from commercial suppliers and used in the subject sensorsand systems. Antibodies in accordance with embodiments of the inventionmay have any suitable binding affinity for an analyte (e.g., anantigen), having a K_(D) value ranging from 10⁻⁴ to 10⁻¹⁵, such as 10⁻⁵,10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³, or 10⁻¹⁴.

Antibody fragments and conjugates may also be used as analyte bindingdomains in the subject surface-associated compositions, and as such,references made herein to the term “antibody” or “antibodies” are to beunderstood as including full length antibodies as well as any fragmentsand/or conjugates thereof. Antibody fragments, for example, include butare not limited to Fv, F(ab), F(ab′), F(ab′)2, and single-chainantibodies having one full-length heavy chain and one full-length lightchain. In some embodiments, an antibody may be a hybrid antibody thatincludes one or more portions of a first antibody that are functionallyattached or connected to one or more portions of a second antibody.Antibody conjugates include, for example, antibodies that are bound toone or more detectable label moieties and/or reporter compounds, suchas, e.g., enzymes, enzyme substrates, radioactive or colorimetriclabels, and the like.

In some embodiments, a surface-associated composition may include one ormore detectable label moieties and/or reporter compounds, such as, e.g.,enzymes, enzyme substrates, radioactive or colorimetric labels, and thelike, in order to facilitate detection of the surface-associatedcomposition.

Crosslinking Compositions

Crosslinking compositions in accordance with embodiments of theinvention are present on the sensor and are configured to crosslink oneanother, as well as one or more surface-associated compositions, in thepresence of an analyte. Crosslinking compositions in accordance withembodiments of the invention include at least two analyte bindingdomains that are configured to specifically bind to an analyte. In someembodiments, a crosslinking composition may include 3, 4, 5, 6, 7, 8, 9or 10 or more analyte binding domains.

Specific binding between an analyte and an analyte binding domainresults in the formation of a stable association between the analyte andthe analyte binding domain. Depending on the nature of the analyte, ananalyte binding domain may include, for example, a member of areceptor/ligand pair; a ligand-binding portion of a receptor; a memberof an antibody/antigen pair; an antigen-binding fragment of an antibody;a hapten; a member of a lectin/carbohydrate pair; a member of anenzyme-substrate pair; biotin/avidin; biotin/streptavidin;digoxin/antidigoxin; a member of a DNA or RNA aptamer binding pair; amember of a peptide aptamer binding pair; a member of ametal/metal-binding peptide pair; a chelating agent; and the like.

In some embodiments, an analyte binding domain of a crosslinkingcomposition includes an antigen. The antigen may specifically bind to ananalyte in a sample, such as an antibody of interest in the sample, or afragment thereof. In some embodiments, an analyte binding domainincludes an antibody, or an antibody fragment. The antibody mayspecifically bind to an analyte of interest in a sample, such as anantigen of interest in the sample.

In some embodiments, a sensor includes a plurality of crosslinkingcompositions whose analyte binding domains recognize different bindingregions of an analyte. As such, in certain embodiments, two or moredifferent analyte binding domains are each configured to bind todifferent portions of the same analyte (e.g., different epitopes on thesame antigen).

In some embodiments, an analyte binding domain of a crosslinkingcomposition includes an antibody. Antibodies in accordance withembodiments of the invention may be monoclonal or polyclonal, and may beany suitable class (isotype), including IgA, IgD, IgE, IgG or IgM.Antibodies in accordance with embodiments of the invention may also beany suitable subclass, including but not limited to IgG1, IgG2, IgG3 orIgG4. Antibodies may be produced by any suitable means, including butnot limited to, inoculation of a suitable mammal with an antigen,followed by recovery and purification of the antibody, or through theuse of recombinant antibody production technology. Antibodies may alsobe obtained from commercial suppliers and used in the subject sensorsand systems. Antibodies in accordance with embodiments of the inventionmay have any suitable binding affinity for an analyte (e.g., andantigen), having a K_(D) value ranging from 10⁻⁴ to 10⁻¹⁵, such as 10⁻⁵,10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³, or 10⁻¹⁴.

Antibody fragments and conjugates may also be used as analyte bindingdomains in the subject crosslinking compositions. Antibody fragments,for example, include but are not limited to Fv, F(ab), F(ab′), F(ab′)2,and single-chain antibodies having one full-length heavy chain and onefull-length light chain. In some embodiments, an antibody may be ahybrid antibody that includes one or more portions of a first antibodythat are functionally attached or connected to one or more portions of asecond antibody. Antibody conjugates include, for example, antibodiesthat are bound to one or more detectable label moieties and/or reportercompounds, such as, e.g., enzymes, enzyme substrates, radioactive orcolorimetric labels, and the like.

In some embodiments, a crosslinking composition may be composed of asingle molecule that has two or more analyte binding domains that areconfigured to specifically bind to an analyte. In some embodiments, acrosslinking composition may include a plurality of different molecules,such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more molecules. In suchembodiments, the molecules that make up the crosslinking composition areconfigured to stably associate with one another to form the crosslinkingcomposition. For example, in some embodiments, a crosslinkingcomposition may include a first molecule with a first analyte bindingdomain, and a second molecule with a second analyte binding domain. Insuch embodiments, the first molecule is configured to stably associatewith the second molecule to form a crosslinking composition.

In some embodiments, a crosslinking composition includes a polypeptidethat has one or more immunoglobulin binding domains that are configuredto form a stable association with an immunoglobulin molecule (e.g., anantibody). Examples of polypeptides that include at least oneimmunoglobulin binding domain include, but are not limited to: proteinA, protein G, protein A/G and protein L. In some embodiments, acrosslinking composition may include a polypeptide molecule with twoimmunoglobulin binding domains, wherein one immunoglobulin molecule(e.g., an antibody) is stably associated with each of the immunoglobulinbinding domains.

In some embodiments, a crosslinking composition may include one or moredetectable label moieties and/or reporter compounds, such as, e.g.,enzymes, enzyme substrates, radioactive or colorimetric labels, and thelike, in order to facilitate detection of the surface-associatedcomposition.

Analytes

The subject sensors may be configured to detect any target analyte ofinterest. Analytes of interest include, but are not limited to, organicand inorganic molecules, environmental pollutants (e.g., heavy metals,pesticides), chemical compounds, therapeutic drugs, drugs of abuse,biomolecules (e.g., hormones, cytokines, proteins, lipids,carbohydrates, nucleic acids), cells, parasites, viruses, bacteria, andfungi (e.g., spores). Analytes of interest also include, but are notlimited to, food borne and other pathogens, such as microbes, e.g.,parasites, bacteria, viruses, fungi, or any toxins produced thereby. Inreference to an analyte that is a microbe, (including a cell, parasite,virus, bacterium or fungus), analytes of interest include any portion ofa microbe (e.g., a molecule or portion thereof that is a part of themicrobe). Non-microbe analytes of interest include, but are not limitedto, molecules or portions thereof that may be produced by a microbe(e.g., a toxin or other molecule that is produced by a microbe).

Bacteria that may be detected using the subject sensors include, but arenot limited to: gram positive bacteria, e.g., Actinomyces, Bacillus,Clostridium, Corynebacterium, Enterococcus, Gardnerella, Lactobacillus,Listeria, Mycobacterium, Mycoplasma, Nocardia, Propionibacterium,Staphylococcus (such as S. aureus, S. epidermidis), Streptococcus (suchas α-hemolytic Streptococcus (e.g., pneumoniae, Viridens), (β-hemolyticStreptococcus (e.g., pyogenes, agalactiae) and γ-hemolytic Streptococcus(e.g., enterococcus)), and Streptomyces; and gram negative bacteria,e.g., Acetobacter, Borrelia, Bortadella, Burkholderia, Campylobacter,Chlamydia, Enterobacter, Eschrichia (such as E. Coli and Salmonella),Fusobacterium, Helicobacter, Haemophilus, Klebsiella, Legionella,Leptospiria, Neisseria, Nitrobacter, Proteus, Pseudomonas, Rickettsia,Salmonella, Serratia, Shigella, Thiobacter, Treponema, Vibrio, andYersinia.

Viruses that may be detected using the subject sensors include, but arenot limited to: double stranded DNA viruses, e.g., Caudoviruses,Herpesviruses, Ligamenviruses, Adenoviruses, Polyomaviruses, andPoxviruses; single stranded DNA viruses, e.g., Anelloviruses,Circoviruses, Nanoviruses and Parvoviruses; double stranded RNA viruses,e.g., Alternaviruses, Chrysoviruses, and Reoviruses; positive-sense (+)single stranded RNA viruses, e.g., Caliciviruses (such as Norovirus),Nidoviruses, Picornaviruses, Tymoviruses, Flaviviruses, Togaviruses, andHepeviruses; negative sense (−) single stranded RNA viruses, e.g.,Mononegaviruses (such as Filoviruses, including Ebola virus and Marburgvirus), Bunyaviruses (such as Hantavirus), Orthomyxoviruses (such asInfluenza virus) and Deltaviruses; single stranded RNA reversetranscriptase (RT) viruses, e.g., Metaviruses, Pseudoviruses andRetroviruses (such as Human Immunodeficiency Virus (HIV)); and doublestranded DNA reverse transcriptase (RT) viruses, e.g., Hepadnaviruses(such as Hepatitis B virus) and Caulimoviruses.

Fungi that may be detected using the subject sensors include:Microsporidia (such as Brachiola, Encephalitozoon, Entercytozoon,Microsporidium, Nosema, Pleistophora, Trachipleistophora, andVittaforma); Chytridiomycota; Blastocladiomycota; Neocallimastigomycota;Glomeromycota; Ascomycota (such as Aspergillus, Candida, Coccidioides,Histoplasma); and Basidiomycota (such as Cryptococcus).

Parasites that may be detected using the subject sensors include, butare not limited to: protozoan organisms (such as Giardia, Malaria,Toxoplasma gondii and Trypanosomes); and helminthes organisms (such astapeworms, roundworms, and flukes).

Examples of non-microbe analytes of interest include, but are notlimited to: Botulinum neurotoxins; Tetanus toxin; Staphylococcal toxins;Alpha toxin; Anthrax toxin; Diptheria toxin; Exotoxin; Pertussis toxin;Shiga toxin and Shiga-like toxin.

Piezoelectric Base

The subject sensors include a piezoelectric base, or substrate. Apiezoelectric base in accordance with embodiments of the inventionincludes a material that produces an electrical charge when a mechanicalstress is imposed on the material, and produces a mechanical stress whenan electrical charge is imposed on the material. The subject sensorsfunction by applying an oscillating electric field to the piezoelectricbase to create a mechanical wave therein. The wave propagates throughthe piezoelectric base and is converted into an electrical signal formeasurement. The resonant frequency of the piezoelectric base can bemeasured, and changes in the resonant frequency resulting from changesin the mechanical properties of the sensor (e.g., from the formation ofcrosslinks between the surface-associated compositions and thecrosslinking compositions in the presence of an analyte) can be utilizedto qualitatively and/or quantitatively determine the amount of ananalyte that is bound to the molecules on the sensor.

A piezoelectric base in accordance with embodiments of the invention mayhave any suitable size and shape. In some embodiments, a piezoelectricbase may be circular, oval, square, rectangular, or hexagonal in shape.In some embodiments, the piezoelectric base may include a texturedsurface. Piezoelectric base components are commercially available invarious forms, such as wafers or discs of suitable sizes and shapes.Commercial suppliers of piezoelectric components and materials include,for example International Crystal Manufacturing Co. Inc. (ICM, Inc.,Oklahoma City, Okla.). In some embodiments, a piezoelectric base mayhave a length dimension that ranges from 2 to 50 mm, such as 5, 10, 15,20, 25, 30, 35, 40 or 45 mm. In some embodiments, a piezoelectric basemay have a width dimension that ranges from 2 to 50 mm, such as 5, 10,15, 20, 25, 30, 35, 40 or 45 mm. In some embodiments, a piezoelectricbase may have a diameter that ranges from 2 to 50 mm, such as 5, 10, 15,20, 25, 30, 35, 40 or 45 mm.

Sensors in accordance with embodiments of the invention may have apiezoelectric base with a thickness that varies, where in some instancesthe thickness ranges from 10 μm to 5 mm, such as 50, 100, 200, 300, 400,500, 600, 700, 800, or 900 μm or more, such as 1, 2, 3 or 4 mm. In someembodiments, the thickness of the piezoelectric base is uniform, e.g.,is the same at each position on the piezoelectric base, while in someembodiments, the thickness of the piezoelectric base is variable, e.g.,is different at different positions on the piezoelectric base.

Piezoelectric base materials in accordance with embodiments of theinvention include, but are not limited to, quartz (SiO₂), berlinite(AlPO₄), gallium orthophosphate (GaPO₄), tourmaline, barium titanate(BaTiO₃), lead zirconate titanate (PZT), zinc oxide (ZnO), aluminumnitride (AiN), polyvinylidene fluoride (PVDF), lithium tantalite(LiTaO₃), lanthanum gallium silicate and potassium sodium tartrate(KNaC₄H₄O₆.4H₂O). In some embodiments, the piezoelectric base is an ATcut quartz crystal. In some embodiments, the piezoelectric base is an SCcut quartz crystal.

In some embodiments, a piezoelectric base may be disposed on or in anon-piezoelectric material. Suitable non-piezoelectric materialsinclude, but are not limited to: polymeric materials (e.g., plastics);metals; glasses; ceramics; or any combination thereof. In someembodiments, a sensor may include a non-piezoelectric material thatstructurally supports the piezoelectric base. In such embodiments, apiezoelectric base may be mounted on a non-piezoelectric material. Forexample, in some embodiments, a piezoelectric base may be mounted on asurface of a non-piezoelectric material. In some embodiments, a sensormay include a non-piezoelectric material having a depression orconcavity therein, and the piezoelectric base may be placed in thedepression or concavity.

Electrodes

Electrodes in accordance with embodiments of the invention may have anysuitable geometry and dimensions, and may be located on thepiezoelectric base to suitably control the application of electricalcharge to, and the detection of electrical charge in, the piezoelectricbase material. For example, in some embodiments, the dimensions of anelectrode may range from 1 μm to 50 mm, such as 10, 20, 30, 40, 50, 60,70, 80 or 90 μm, or such as 0.1, 1, 10, 20, 30 or 40 mm. The geometryand dimensions of an electrode may be varied in order to conform to theshape of the piezoelectric base material. For example, in someembodiments, an electrode may include a portion having a rectangularshape, and/or an arced, circular, or semi-circular shape.

In some embodiments, an electrode may be formed into an interdigitatedstructure, meaning that the electrode includes a first and secondplurality of comb-like projections that are configured to interlock withone another to form a zipper-like pattern. An electrode with aninterdigitated structure can be configured to convert an electricalsignal into an acoustic wave that propagates through the pieozoelectricbase, and vice versa. In some embodiments, a sensor may include a firstelectrode with an interdigitated structure that functions as an inputelectrode that converts an electrical signal into a mechanical wave thatpropagates through the piezoelectric base material, and a secondelectrode with an interdigitated structure that functions as an outputelectrode that converts a mechanical wave into an electrical signal.

Electrodes in accordance with embodiments of the invention may includeany conductive material, including but not limited to, aluminum, carbon,chromium, cobalt, copper, molybdenum, nickel, palladium, platinum,silicon, silver, tin oxide, titanium, tungsten, zinc, or gold.

Electrodes in accordance with embodiments of the invention areconfigured to induce mechanical oscillations in the piezoelectricmaterial when an appropriate current or voltage is applied to theelectrode. In response to the applied current or voltage, thepiezoelectric base is configured to vibrate at a resonant frequency. Insome embodiments, the resonant frequency of the piezoelectric baseranges from 0.1-100 Hz, such as 25-75 Hz. In some embodiments, theresonant frequency of the piezoelectric base ranges from 1-100 kHz, suchas 25-75 kHz. In some embodiments, the resonant frequency of thepiezoelectric base ranges from 1-30 MHz, such as 10-15 MHz.

The application of a suitable electrical signal (e.g., a current orvoltage) to an electrode creates a standing shear wave in thepiezoelectric material, and the characteristics of the standing shearwave can be detected and measured using standard electrical circuitryand data recording devices. The frequency of oscillation of the sensoris partially dependent on the thickness of the piezoelectric basematerial, and changes in the thickness of the base material or itsmechanical properties (e.g., the dynamic modulus of the sensor)correlate directly to one or more changes in the oscillation frequency,and/or the parameters of an oscillator circuit that is used to drive thesensor at a resonant frequency. For example, the binding of an analyteto the surface-associated compositions and crosslinking compositionsresults in the formation of crosslinks between the compositions on thesensor, which changes the dynamic modulus of the sensor and modulatesthe oscillation frequency of the sensor. Any changes in the resonantfrequency of the sensor (e.g., changes in the frequency, the amplitudeand/or the frequency bandwidth of the resonant frequency), and/or theparameters of the oscillator circuit that is used to drive the sensor ata resonant frequency, are measured using standard techniques, and thedata is used to determine the amount of the analyte bound to the sensor.Various characteristics of the frequency change can be quantified andcorrelated precisely to the mass change of the piezoelectric base usingSauerbrey's equation (Δm=−C·Δf, where Δm is the change in mass, Δf isthe change in frequency, and −C is a constant that is based on theresonant frequency, the piezoelectrically active area of thepiezoelectric base, the density of the piezoelectric base material, andthe shear modulus of the piezoelectric base material), therebyfacilitating the quantification of the amount of analyte bound to thesensor. Aspects of the resonant frequency and standing shear wave in thepiezoelectric base that can be measured include, but are not limited to,the frequency, the amplitude, and the frequency bandwidth of thewaveform that is generated in the piezoelectric base.

Sensors in accordance with embodiments of the invention may include astructure that is configured to retain a liquid sample in contact withthe sensor. For example, in some embodiments, a sensor may include awell that includes a walled structure that is configured to hold avolume of liquid. A well in accordance with embodiments of the inventioncan have any suitable shape, and may have, e.g., a square, rectangular,circular, oval, or hexagonal cross-sectional shape when viewed fromabove. Wells in accordance with embodiments of the invention may have adepth that varies, and in some instances may range from 1, 2, 3, 4, 5,6, 7, 8, 9 or 10 mm deep or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 cm deep or more. Wells in accordance with embodiments of theinvention may have a length, a width, or a diameter that varies, and insome instances may range from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cm ormore.

In some embodiments, the walls of a well may be substantiallyperpendicular to the bottom of the well. In certain embodiments, thewalls of a well may be positioned at an angle with respect to the bottomof the well, wherein the angle may range from 80 degrees to 45 degrees,such as 50 to 70 degrees. In some embodiments, the walls of a well maybe straight. In some embodiments, the walls of a well may be curved orflared, such that the diameter of the well increases or decreases in thevertical direction. In some embodiments, a well may be configured to beoperatively attached or coupled to a sensor in order to facilitateretaining a liquid sample in contact with the sensor.

In some embodiments, a well is configured to hold a volume of liquidranging from 10 μL to 10 mL, such as 50, 100, 250, 500, or 750 μL ormore, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9 mL or more. In someembodiments, a well is configured such that the piezoelectric base ofthe sensor is disposed at the bottom of the well. In some embodiments, awell is configured such that the piezoelectric base is disposed on aside of the well (e.g., on an inner surface of a wall of the well).

In some embodiments, a sensor may include a depression or concavity thatis configured to retain a liquid sample in contact with the sensor. Adepression in accordance with embodiments of the invention can have anysuitable shape, and may have, e.g., a square, rectangular, circular,oval, or hexagonal cross-sectional shape when viewed from above. Adepression in accordance with embodiments of the invention may have adepth that varies, and in some instances may range from 1, 2, 3, 4, 5,6, 7, 8, 9 or 10 mm deep or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 cm deep or more. In some embodiments, a depression may have a length,a width, or a diameter that varies, and in some instances may range from1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cm or more. In some embodiments, adepression is configured to hold a volume of liquid ranging from 100 μLto 10 mL, such as 1 to 5 mL. In some embodiments, a depression isconfigured such that the piezoelectric base of the sensor is disposed atthe bottom of the depression. In some embodiments, a depression isconfigured such that the piezoelectric base is disposed on a side of thedepression.

In some embodiments, a sensor may include an extended portion (having adistal end and a proximal end) that is configured to allow the sensor tobe dipped into or immersed in a liquid sample so that the sample cancontact the sensor. For example, in some embodiments, a sensor mayinclude an extended portion having a length that may range from 5 to 500cm, such as 25 to 250 cm and a width that may range from 1 to 10 cm,such as 2 to 5 cm. In some embodiments, the extended portion may berigid, while in some embodiments, the extended portion may be flexibleand configured such that an operator can bend or shape the extendedportion into a desired shape for use in contacting a sample. Forexample, in some embodiments, a sensor includes an extended portion thatcan be bent or curved by an operator to facilitate contacting the sensorwith a sample. In such embodiments, the extended portion is configuredsuch that the piezoelectric base of the sensor is disposed at the distalend of the extended portion, and is operatively connected to the otherparts of the sensor. An extended portion of a sensor in accordance withembodiments of the invention can be made from any suitable material,such as plastic, metal, glass or ceramic, or any suitable combinationthereof.

In some embodiments, the subject sensors include a computer-readablemedium (e.g., an EPROM chip (erasable programmable read-only memorychip) that is configured to store data). The stored data may includeinformation regarding a calibration value or parameter for the sensor,and/or an operating value or parameter for the sensor. When the sensoris coupled to a detection unit, as described further below, the datastored on the computer-readable medium of the sensor can be accessed bythe detection unit and used during operation of the sensor. In someembodiments, a sensor may include a computer-readable medium thatcontains data relating to one or more calibration values for the sensor,or one or more calibration values for a particular manufacturing lot ofsensors. In some embodiments, the computer-readable medium on the sensormay include data relating to the type and/or class of analyte that canbe detected by the sensor, the type of surface-associated and/orcrosslinking compositions on the sensor, the type of detectable label onthe sensor, or any other useful information relating to the operationand use of the sensor with the subject systems and methods.

In some embodiments, the computer-readable medium on the sensor mayinclude information that relates to an analyte signature. By “analytesignature” is meant a specific set of values that is generated by thesensor when a specific analyte binds to the sensor. In some embodiments,information relating to an analyte signature may be stored on thecomputer-readable medium of the sensor, and may be accessed by thedetection unit when the sensor is coupled to the detection unit. Theanalyte signature information can then be used to identify and quantifythe amount of an analyte in a test sample that is contacted with thesensor.

In some embodiments, a detection unit, as described further below, mayinclude a computer-readable medium that is configured to store data. Thestored data may include information regarding a calibration value orparameter for a particular sensor, and/or an operating value orparameter for a particular sensor. In some embodiments, the detectionunit is configured to obtain identification information from the sensor(e.g., a particular lot number or other identifier) and to use theidentification information when operating the sensor.

Sensors in accordance with embodiments of the invention may includeadditional components that are configured to facilitate the operativeconnection of the sensor to a detection unit, as described furtherbelow. In some embodiments, a sensor may include one or more electricalleads and/or electrode contacts that are configured to establish anelectrical connection between the sensor and the detection unit.Electrical leads and/or electrode contacts may have any suitablegeometry and/or dimensions as required to establish the necessaryelectrical contact between the sensor and the detection unit. In someembodiments, a sensor may include mechanical elements that areconfigured to connect with a detection unit to facilitate the operativeconnection of the sensor to the detection unit. For example, in someembodiments, a sensor may include a clip, a clasp, a snap-fit element,or any other suitable mechanical component that is configured to engagewith a corresponding mechanical component on the detection unit in orderto operatively couple the sensor to the detection unit. In someembodiments, a sensor and/or a detection unit may further include arelease component that is configured to dis-engage the sensor from thedetection unit. For example, in some embodiments, a detection unit mayinclude a button or a lever that can be used to dis-engage a sensor fromthe detection unit.

Referring now to FIG. 1, an embodiment of a subject sensor that includesa plurality of surface-associated compositions and crosslinkingcompositions is depicted. In the depicted embodiment, the piezoelectricbase 1 is shown, as well as a plurality of surface-associatedcompositions 2 and a plurality of crosslinking compositions 3. Thedepicted surface-associated compositions 2 and crosslinking compositions3 are configured to crosslink one another in the presence of an analyte.

Methods of Making Sensors

Aspects of the invention include methods of making the subject sensors,as described above. In some embodiments, a sensor is made by contactinga piezoelectric base (or an electrode formed thereon) with a compositionunder conditions that facilitate the formation of a stable associationbetween the composition and a surface of the piezoelectric base. Forexample, in some embodiments, a liquid that includes a plurality ofsurface compositions is contacted with the piezoelectric base, and thesurface compositions stably associate with the piezoelectric base toform a plurality of surface-associated compositions on the piezoelectricbase.

Contacting the piezoelectric base with a liquid that includes thesurface compositions can be accomplished by any suitable method,including, but not limited to: immersing the piezoelectric base in theliquid; depositing the liquid on top of the piezoelectric base using,e.g., a pipette or micropipette; spraying the liquid onto thepiezoelectric base, spin coating the liquid onto the piezoelectric base,etc.

The liquid that includes the plurality of surface compositions isconfigured to maintain the surface compositions under suitableconditions to facilitate their stable association with the piezoelectricbase. As such, depending on the nature of the surface compositions, theliquid may be an aqueous or non-aqueous liquid, may contain any suitablebuffering components, may have any suitable pH, and may be maintained atany suitable temperature that does not degrade the surface-associatedcompositions. In some embodiments, the surface-associated compositionsinclude a polypeptide (e.g., an antibody), and the liquid is an aqueousbuffer, such as water or phosphate buffered saline (PBS), having a pHthat ranges from 4 to 8, such as 6 to 7, and a temperature that rangesfrom 5 to 20 degrees C. Any suitable amount of surface compositions maybe present in the liquid. In some embodiments, the concentration of thesurface compositions in the liquid ranges from 0.1 μg/mL to 10 mg/mL,such as 1 μg/mL, 100 μg/mL, 500 μg/mL, or more, such as 1 to 5 mg/mL.

After the liquid is contacted with the piezoelectric base, the liquid isallowed to evaporate, leaving a plurality of surface-associatedcompositions on the surface of the piezoelectric base.

Once the surface-associated compositions have stably associated with thepiezoelectric base, a plurality of crosslinking compositions isdeposited on top of the surface-associated compositions. For example, insome embodiments, a liquid that includes a plurality of crosslinkingcompositions is contacted with the sensor, and the liquid is evaporatedto leave a plurality of crosslinking compositions on the sensor.

Contacting the sensor with the liquid that includes the crosslinkingcompositions can be accomplished by any suitable method, including, butnot limited to: immersing the sensor in the liquid; depositing theliquid on top of the sensor using, e.g., a pipette or micropipette;spraying the liquid onto the sensor, spin coating the liquid onto thesensor, etc.

The liquid that includes the plurality of crosslinking compositions isconfigured to maintain the crosslinking compositions under suitableconditions to facilitate their deposition on the sensor. As such,depending on the nature of the crosslinking compositions, the liquid maybe an aqueous or non-aqueous liquid, may contain any suitable bufferingcomponents, may have any suitable pH, and may be maintained at anysuitable temperature that does not degrade the crosslinkingcompositions. In some embodiments, a crosslinking composition includes apolypeptide (e.g., an antibody), and the liquid is an aqueous buffer,such as water or phosphate buffered saline (PBS), having a pH thatranges from 4 to 8, such as 6 to 7, and a temperature that ranges from 5to 20 degrees C. Any suitable amount of crosslinking compositions may bepresent in the liquid. In some embodiments, the concentration of thecrosslinking compositions in the liquid ranges from 0.1 μg/mL to 10mg/mL, such as 1 μg/mL, 100 μg/mL, 500 μg/mL, or more, such as 1 to 5mg/mL.

After the liquid is contacted with the sensor, the liquid is allowed toevaporate, leaving a plurality of crosslinking compositions deposited ontop of the surface-associated compositions on the sensor. The sensor isthen ready for use, as provided in greater detail herein.

In some embodiments, a sensor can be made by adding activated carbon toone or more of the solutions that are deposited on the surface. Forexample, in some embodiments, activated carbon is combined with theliquid that includes the plurality of surface compositions. In someembodiments, activated carbon is combined with the liquid that includesthe plurality of crosslinking compositions.

In some embodiments, a sensor is made by depositing a plurality of firstmolecules on a surface of the piezoelectric base, and then depositing aplurality of second molecules on top of the plurality of firstmolecules. The first and the second molecules are members of both thesurface-associated compositions and the crosslinking compositions thatare to be formed on the sensor. Following their deposition on thesensor, the first and second molecules self-assemble into a plurality ofsurface-associated compositions that are stably associated with thepiezoelectric base, and a plurality of crosslinking compositions.

For example, in some embodiments, a liquid that includes a plurality offirst molecules (e.g., protein G molecules) is contacted with thepiezoelectric base, and the first molecules stably associate with asurface of the piezoelectric base. The liquid includes an excess amountof the first molecules, such that the entire surface of thepiezoelectric base that is available for stable association becomescovered with the first molecules. The excess first molecules remainpresent on the sensor, but are not stably associated with a surface ofthe piezoelectric base.

In some embodiments, activated carbon is combined with the liquid thatincludes the plurality of molecules. In some embodiments, activatedcarbon is combined with the liquid that includes the plurality of secondmolecules.

After the liquid is contacted with the piezoelectric base, the liquid isallowed to evaporate, leaving a plurality of the first molecules stablyassociated with the surface of the piezoelectric base, and a pluralityof the first molecules present on the sensor, but not stably associatedwith the piezoelectric base.

Next, a liquid that includes a plurality of second molecules (e.g., aplurality of polyclonal antibodies) is deposited on top of the pluralityof first molecules. A portion of the second molecules stably associatewith the first molecules that are already stably associated with thesurface of the piezoelectric base, thereby forming a plurality ofsurface-associated compositions that include a first molecule (e.g., aprotein G molecule) and a second molecule (e.g., a polyclonal antibody).Another portion of the second molecules stably associate with the firstmolecules that are not stably associated with the surface of thepiezoelectric base, thereby forming a plurality of crosslinkingcompositions that include a first molecule (e.g., a protein G molecule)and at least one second molecule (e.g., a polyclonal antibody). Theliquid that includes the plurality of second molecules is allowed toevaporate, leaving a plurality of surface-associated compositions thatare stably associated with a surface of the piezoelectric base, and aplurality of crosslinking compositions on the sensor.

Sensors in accordance with embodiments of the invention can be mademanually, using the processes described above, or can be made usingautomated or semi-automated equipment. In some embodiments, a pluralityof sensors can be made using automated or semi-automated equipmentoperating in a “batch” mode, wherein a batch of sensors are made at thesame time, or in a continuous mode, wherein sensors are continuouslyproduced.

In some embodiments, methods of making the subject sensors includedepositing or forming one or more electrodes on the surface of thepiezoelectric base. Deposition of the electrodes can be accomplishedusing any suitable method, including, e.g., photolithography techniques,vapor deposition techniques, electrode printing techniques, and thelike.

In one preferred embodiment, a sensor comprises surface-associatedcompositions that are made from protein G and polyclonal antibodies, andcomprises crosslinking compositions that are also made from protein Gand polyclonal antibodies.

In another preferred embodiment, a sensor comprises surface-associatedcompositions that are made from protein A and polyclonal antibodies, andcomprises crosslinking compositions that are also made from protein Aand polyclonal antibodies.

In another preferred embodiment, a sensor comprises surface-associatedcompositions that are made from protein G and monoclonal antibodies, andcomprises crosslinking compositions that are also made from protein Gand monoclonal antibodies.

In another preferred embodiment, a sensor comprises surface-associatedcompositions that are made from protein A and monoclonal antibodies, andcomprises crosslinking compositions that are also made from protein Aand monoclonal antibodies.

Systems

Aspects of the invention include systems that can be used in connectionwith the subject sensors to carry out the methods described herein.Systems in accordance with embodiments of the invention includedetection units that are configured to interact with a subject sensor,as well as peripheral components that find use in carrying out thesubject methods.

In some embodiments, a system includes a detection unit that isconfigured to connect to one or more sensors. In some embodiments, adetection unit includes a connection feature or docking port thatoperatively connects a sensor to the detection unit. In someembodiments, the connection feature includes electrical leads and/orelectrode contacts that are configured to establish an electricalconnection between the detection unit and one or more features of thesensor. In some embodiments, a detection unit may include a plurality ofconnection features or docking ports, such that a plurality of sensorscan be connected simultaneously to the detection unit. In someembodiments, a detection unit may include a number of docking portsranging from 1 to 10, such as 2, 3, 4, 5, 6, 7, 8 or 9. In someembodiments, a sensor is permanently attached to the detection unit. Insome embodiments, a detection unit may include a release component thatis configured to dis-engage a sensor from the detection unit. Forexample, in some embodiments, a detection unit may include a button or alever that can be used to dis-engage a sensor from the detection unit.

In some embodiments, a system includes one or more temperature controlelements that are configured to control the temperature of one or moreportions of the sensor and/or a sample this being contacted with thesensor. For example, in some embodiments, a system includes atemperature controller that is configured to maintain the sensor withina target temperature range. In some embodiments, a detection unitincludes a temperature controller that is configured to maintain asample, or a portion thereof, at a target temperature for analysis.Temperature control elements in accordance with embodiments of thedevice may include temperature blankets, resistive heaters,thermoelectric heaters or coolers, fans, and the like.

In some embodiments, a system includes an oscillator circuit that isconfigured to electrically connect to a sensor. The oscillator circuitis configured to drive the sensor at one or more frequencies, includingbut not limited to, a resonant frequency of the piezoelectric basematerial of the sensor. In certain embodiments, the oscillator circuitincludes an automatic gain control (AGC) portion. Oscillator circuits inaccordance with embodiments of the invention may include one or moreresistors, capacitors and/or inductors, arranged in series and/or inparallel. Aspects of the automatic gain control portion of theoscillator circuit that can be measured include, but are not limited to,the voltage, resistance, admittance, impedance or conductance values ofthe circuit. In some embodiments, an oscillator circuit is a leveroscillator that is designed for use with liquid applications.

In some embodiments, a system includes a comparator/receiver componentthat is configured to receive a signal from a sensor, and is alsoconfigured to receive a signal from an oscillator circuit that iselectrically connected to the sensor. The comparator/receiver componentis configured to compare and measure one or more features of the signalsthat are received from the sensor and the oscillator circuit. Themeasured feature(s) of the sensor and the oscillator circuit can then beused to determine the concentration of the analyte that is present onthe sensor.

In some embodiments, a system includes a frequency spectrum analyzerthat is configured to measure one of more features of the sensor as afunction of the oscillation frequency applied to the sensor. Forexample, in some embodiments, a frequency spectrum analyzer isconfigured to measure a change in the frequency, amplitude and/orfrequency bandwidth of a waveform in the sensor as a function of theoscillation frequency that is applied to the sensor by the oscillatorcircuit. The data acquired by the frequency spectrum analyzer isrecorded and analyzed by the system.

In some embodiments, a system includes a controller that is configuredor adapted to control or operate one or more components of the subjectsystems. In some embodiments, the controller is in communication withone or more components of the system and is configured to controlaspects of the system and/or execute one or more operations or functionsof the system. In some embodiments, a system includes a processor and acomputer-readable medium, which may include memory media and/or storagemedia. Applications and/or operating systems embodied ascomputer-readable instructions on computer-readable memory can beexecuted by the processor to provide some or all of the functionalitiesdescribed herein, including by not limited to, carrying out one or moreof the method steps described herein, acquiring and processing dataobtained from the subject sensors and/or systems, and/or applying one ormore algorithms or other manipulations to the data for analysis.

In some embodiments, a system includes a user interface, such as agraphical user interface (GUI), that is adapted or configured to receiveinput from a user. In some embodiments, a GUI is configured to displaydata or information to a user. In some embodiments, the subject systemsinclude an indicator or readout screen that can be used to communicateinformation to a user. For example, in some embodiments, a system mayinclude an indicator light that is illuminated when the system detectsan analyte of interest in a sample above an established concentrationthreshold. In some embodiments, a readout screen is configured todisplay a list of one or more analytes that are detected in a sample.

Additional aspects of the subject systems may include, but are notlimited to, analog to digital converters that are configured to convertone or more continuous physical quantities, such as a voltagemeasurement, to a digital number that represents the quantity'samplitude. Aspects of the subject systems also include data exchangefeatures, such as, e.g., USB ports, Ethernet ports, or other data portsthat are configured to establish a connection that can be used toexchange/transmit data between two or more components of the system.Aspects of the subject systems also include wireless transmissioncomponents, such as WiFi components, that are configured to wirelesslytransmit data between two or more components of the system.

Aspects of the subject systems may also include computer processors,data storage, and/or database components that can be used to storeand/or analyze data that is acquired by the subject systems. Suchcomponents can be physically connected to other components of thesubject systems, such as, e.g., via a USB connection, or can beconfigured to wirelessly communicate with other components of thesubject systems, e.g., via WiFi connection, or via the Internet. In someembodiments, computer processors, data storage and/or databasecomponents of the subject systems may be remotely located, e.g., may belocated at a physical location that is different from the physicallocation of the sensor and/or the detection unit.

Aspects of the subject systems may also include power components, suchas batteries and/or power cables that are configured to provideelectrical power to the subject sensors and systems. Power components inaccordance with embodiments of the invention may be modular and may beconfigured to be removeably coupled to the subject systems for purposesof providing power thereto, for example, one or more batteries orbattery packs that are configured to be inserted into or otherwisecoupled to the subject systems. In some embodiments, the subject systemsinclude power cables that are configured to establish electrical contactwith standard power outlets.

In some embodiments, the various features of the subject systems areformed into a single device that includes a housing formed from suitablematerials, such as plastic, metal, glass or ceramic materials, and anycombinations thereof. For example, in some embodiments, a detection unitis formed from a plastic housing, and various additional components ofthe system are located within the housing. In some embodiments, a systemis formed into a single bench-top device that can be used to carry outthe subject methods, as described further below. In some embodiments, asystem is formed into a single, hand-held device that can be carried bya user.

In some embodiments, various features of the subject systems can beformed into two or more separate devices, and the devices can beconfigured to transmit data and/or operating parameters between oneanother. For example, in some embodiments, a sensor unit that includes ahousing, a sensor, an oscillator circuit, a controller, and a processoris configured to interact with a detection unit that includes a housing,a processor, and a comparator/receiver component. In some embodiments,the sensor unit is configured to communicate wirelessly with thedetection unit, while is some embodiments, the sensor unit may beconnected to the detection unit via, e.g., a USB cable. In use, thesensor unit collects data from a sample and transmits the data to thedetection unit, where the data is analyzed and evaluated to determinewhether a target analyte is present in the sample. In some embodiments,the detection unit transmits data and/or operating parameters to thesensor unit.

In some embodiments, the subject systems include a plurality of sensorunits that can be located at different locations, e.g., forenvironmental monitoring. For example, in some embodiments, a pluralityof sensor units can be placed at various geographical locations, and canbe used to monitor the presence of an analyte in a sample collected fromeach location. Data can be transmitted from the plurality of sensorunits to one or more detection units, where the data is analyzed. Insome embodiments, each of the sensor units may include a plurality ofsensors, e.g., a sensor array, wherein each sensor is configured todetect a different target analyte, so that a sample can be analyzed,using the sensor array, for the presence of a plurality of targetanalytes. In certain embodiments, a system may include a “blank” sensor,or a negative control sensor, that is configured to provide a negativecontrol for analysis. In some embodiments, a system may include apositive control sensor that is configured to provide a positive controlfor analysis.

Aspects of the subject systems may include activated carbon compositionsthat can be used to concentrate an analyte or antigen in a sample.Activated carbon compositions in accordance with embodiments of theinvention include activated carbon particulates, granules, powders,filaments and nanotubes. In use, an activated carbon composition iscontacted with a sample that contains an analyte of interest. Theanalyte is adsorbed onto the activated carbon composition, therebycreating a multi-valent composition that includes multiple analytemolecules that can bind to the molecules on the sensor. Accordingly,exposing a sample to an activated carbon composition can be used toconcentrate the analyte onto the carbon composition, and thereby amplifythe signal that is detected by the sensor. In some embodiments, anactivated carbon composition is present on a surface of a sensor (i.e.,is disposed on a surface of a sensor). In some embodiments, an activatedcarbon composition can be deposited on a surface of a sensor during aproduction process. For example, in some embodiments, an activatedcarbon composition is combined with one or more components to bedeposited on a sensor surface (e.g., combined with a solution thatcomprises a plurality of surface compositions and/or crosslinkingcompositions) and deposited on the surface of the sensor. In someembodiments, an activated carbon composition can be deposited on asurface of a sensor after a production process. For example, in someembodiments, an activated carbon composition is deposited on a surfaceof a sensor after the surface-associated compositions and crosslinkingcompositions have been deposited.

Aspects of the subject systems may include secondary detection devicesthat are configured to detect and/or quantify one or more detectablelabels or moieties that may be present on the compositions of thesubject sensors. Such secondary detection devices include, but are notlimited to, spectrophotometers, fluoroscopes, ellipsometers, and thelike. After a subject sensor is contacted with a sample that contains atarget analyte, the analyte binds to the compositions on the sensor andcreates a crosslinked network of molecules. The presence of the analyteon the sensor is detected by the subject systems, as described above.For purposes of verifying the results obtained from the sensor, thesubject secondary detection devices can be used to confirm the presenceof the analyte in the sample by further analyzing the sensor.

When a crosslinked network of molecules is formed on the sensor as aresult of a target analyte binding to the sensor, the surface-associatedcompositions and crosslinking compositions are bound in place on thesensor. As described above, in some embodiments, the compositions mayinclude a detectable label. The subject secondary detection devices canbe used to quantify the amount of the detectable label that is presenton the sensor, thereby verifying the results obtained from the sensor.In certain embodiments, a sensor may include a cap element that isconfigured to be removeably coupled to the sensor. The cap is configuredto be removed from the sensor so that the sensor can be contacted with asample. Following detection of an analyte in the sample, the cap can bereplaced in order to protect the sensor tip. The sensor can then beanalyzed by a secondary detection device to confirm the results of thesample analysis. For example, in some embodiments, the cap can beremoved from the tip of a used sensor, and the used sensor can beanalyzed with a secondary detection device to confirm the results of theanalysis by confirming the amount of a detectable label that is presenton the sensor, thus indicating the amount of the analyte that waspresent in the sample.

FIG. 5 shows a schematic representation of a subject sensor 10 that iselectrically connected to a system 11 that includes electrical circuitryfor driving the sensor and collecting data from the sensor. The system11 includes an oscillator 12 that is configured to drive the sensor 10at a plurality of different frequencies. The system 11 also includes acomparator/receiver 13 that is configured to receive one or more signalsfrom the oscillator 12 and one or more signals from the sensor 10, andto compare the received signals to each other. The system 11 alsoincludes a processor 14 that includes a computer readable medium that isconfigured to receive data from the sensor 10 and from thecomparator/receiver 13, and to process, store and/or transmit the dataas needed. The system 11 also includes an indicator/readout screen 15that is configured to receive a signal from the processor 14 and todisplay that signal to a user. The system 11 also includes a controller16 that is configured to control one or more portions of a testprocedure. The system 11 also includes a data transmission component 17that is configured to transmit data outside the system 11. Finally, thesystem 11 also includes a power supply 18 that is configured to supplypower to the system 11.

FIG. 6 shows a schematic representation of a signal received from asubject sensor as a function of time after the sensor has been contactedwith a sample containing an analyte. The signal is plotted to show theamplitude (dB) as a function of both time (s) and frequency (MHz). Asshown, the change in frequency (Δf) can be measured as a function oftime after a sample containing an analyte is contacted with the sensor.

Depicted in FIG. 7 is a system that includes a sensor and a detectioncomponent with a data exchange feature that can be connected to acomputer. As shown, the sensor includes a piezoelectric base (coatedquartz crystal) and a computer-readable medium (EPROM). The depicteddetection component includes a lever oscillator circuit, frequencycounter circuit, wireless communication circuit, LCD screen and supportcircuit, and a USB port circuit and connector for communicating with acomputer. FIG. 8 depicts the features and functionality of a computerthat is connected to the system depicted in FIG. 7. As shown in FIG. 8,the computer is connected to the Internet, and can interface withcloudware to access various types of information, including but notlimited to, a database of all sensors (test cartridges, TCs), testresults, data subscription registration information, test algorithms,calibration information, as well as computer software updates.

Methods of Use

Methods in accordance with embodiments of the invention may be used todetect an analyte in a sample. The subject methods involve contacting asensor, as described above, with a sample that is suspected ofcontaining an analyte of interest, and detecting the presence or absenceof the analyte in the sample.

An analyte that is present in a sample binds to the compositions thatare present on the sensor and forms crosslinks between the compositions.This crosslinking changes the mechanical properties of the sensor (e.g.,changes the dynamic modulus of the sensor). A voltage or current isapplied to an oscillator circuit that is electrically connected to thesensor. The oscillator circuit drives the sensor at one or morefrequencies, and changes in the mechanical properties of the sensorresulting from crosslinking of the surface-associated compositions andcrosslinking compositions are detected. In some embodiments, changes inthe sensor and/or the parameters of the oscillator circuit are measuredas a function of time. The magnitude and type of changes in the sensorand/or oscillator circuit are used to determine the concentration of theanalyte in the sample.

Methods in accordance with embodiments of the invention can be used toanalyze any sample that contains, or may contain, an analyte ofinterest. In some embodiments, a sample is a liquid sample. Liquidsamples that are amenable to analysis with the subject sensors andsystems include aqueous as well as non-aqueous liquids. In someembodiments, a liquid sample may be a bodily fluid, including but notlimited to: amniotic fluid, aqueous or vitreous humor, bile, blood,breast milk, cerebrospinal fluid, cerumen (ear wax), lymphatic fluid,mucus, pleural fluid, pus, saliva, semen, sputum, synovial fluid, sweat,tears, urine, or vaginal fluid or secretions. In some embodiments, asample is a non-liquid sample, e.g., a solid or semi-solid composition.

In some embodiments, the subject methods do not involve preparing asample for analysis. For example, in certain embodiments, the subjectmethods involve contacting a sample with a subject sensor withoutpreparing or modifying the liquid sample in any way. In someembodiments, the subject methods involve contacting a body fluid with asubject sensor without preparing or modifying the body fluid in any way.

In some embodiments, the subject methods involve preparing a sample foranalysis by combining the sample with one or more reagents. For example,in some embodiments, a liquid sample may be diluted by combining thesample with an appropriate diluent. Similarly, in some embodiments, asolid or semi-solid sample may be combined with a liquid in order tocreate a liquid sample that includes the solid or semi-solid sampleeither in suspension or in solution. In certain embodiments, otherreagents may be added to a sample in order to facilitate the analysis ofan analyte in the sample, such as emulsifiers, chelating reagents, andthe like. In some embodiments, an activated carbon composition may becombined with the sample in order to collect one or more molecules ofthe analyte on the surface of the activated carbon composition. Incertain embodiments, the activated carbon composition includes a carbonnanotube.

In some embodiments, the methods involve mixing a liquid sample with oneor more reagents and/or diluents to prepare the sample for analysis. Forexample, in some embodiments, a liquid sample may be mixed with adiluent to reduce the concentration of an analyte in the sample. Variousdilution techniques can be used in conjunction with the subject methods,including but not limited to, diluting a sample by a specific dilutionfactor, or performing a serial dilution of the sample.

Contacting a sensor with a sample can be accomplished using any suitabletechnique. For example, in some embodiments, a sensor may be immersed ina liquid sample, e.g., a sensor can be immersed in a body of water, orimmersed in a container that contains a liquid sample of interest. Insome embodiments, a liquid sample can be collected in a suitablecontainer, and a sensor is then inserted into the container to contactthe sample. In certain embodiments, a container for sample collectionmay be configured to be sealed after a sample is placed inside in orderto maintain sterility of the sample or to prevent adulteration of thesample, and also to protect a technician or operator from contacting thesample. In certain embodiments, a sensor may be configured to fluidlyconnect to a container that holds a liquid sample, thereby contactingthe liquid sample with the sensor. In some embodiments, a liquid samplecan be collected using a suitable device, e.g., a pipette ormicropipette, and the liquid sample can then be deposited directly ontothe sensor.

In some embodiments, the methods involve contacting a substantially drysensor with a sample to be analyzed. In such embodiments, the samplehydrates a surface of the sensor. In some embodiments, the methodsinvolve hydrating a surface of sensor with a liquid that does notcontain a target analyte (e.g., a saline solution), and then contactingthe sensor with a sample to be analyzed. For example, in certainembodiments, the methods involve contacting a surface of a sensor with asaline solution to hydrate the sensor, and then contacting the surfaceof the sensor with a sample that contains (or is suspected ofcontaining) a target analyte.

Aspects of the subject methods involve driving a sensor at one or morefrequencies using an oscillator circuit, as described above, andmeasuring one or more parameters of the sensor and/or the oscillatorcircuit as a function of time at each frequency after the sensor hasbeen contacted with a sample. In some embodiments, the subject methodsinvolve measuring the frequency of a waveform that is generated in thesensor in response to an input voltage or current that is applied to thesensor by an oscillator circuit. In some embodiments, the subjectmethods involve measuring the amplitude of the waveform in the sensor.In certain embodiments, the subject methods involve measuring thefrequency bandwidth, or Q-factor, of the waveform in the sensor. Anycombination of the frequency, amplitude and/or frequency bandwidth of awaveform in the sensor may be measured and used in the subject methodsfor determining the concentration of an analyte in a sample that isapplied to the sensor. In certain embodiments, one or more of thefrequency, amplitude or frequency bandwidth of a waveform in the sensoris measured as a function of time. In some embodiments, one or more evenor odd harmonics of a fundamental test frequency are measured.

Aspects of the subject methods involve measuring one or more parametersof an oscillator circuit that drives the sensor at one or morefrequencies. As reviewed above, oscillator circuits in accordance withembodiments of the invention may include an automatic gain controlportion, and may include one or more resistors, capacitors and/orinductors, arranged in series and/or in parallel. Aspects of the subjectmethods may include measuring a voltage, resistance, admittance,impedance or conductance value of an oscillator circuit, or any portionthereof, such as an automatic gain control portion of the oscillatorcircuit. Any combination of the voltage, resistance, admittance,impedance or conductance values of the oscillator circuit can bemeasured and used in the subject methods for determining a concentrationof an analyte in a sample that is applied to the sensor. In someembodiments, one or more of the voltage, resistance, admittance,impedance or conductance values of the oscillator circuit is measured asa function of time.

In some embodiments, the subject methods involve applying a drivingvoltage or current (e.g., an alternating current) to a sensor for aperiod of time, and then measuring one or more parameters of the sensoras a function of time once the driving voltage or current is shut off.In such embodiments, the decay rate or dissipation of energy in thesensor can be measured as a function of time, and can be used todetermine the concentration of an analyte in the sample that was appliedto the sensor.

In some embodiments, the subject methods involve using a frequencyspectrum analyzer to analyze one or more attributes of a waveform (e.g.,the frequency, amplitude and/or frequency bandwidth) generated in thepiezoelectric base as a function of one or more electronic signals thatare applied to the sensor by an oscillator circuit. For example, in someembodiments, an oscillator circuit is used to drive the sensor at aplurality of different frequencies, and a frequency spectrum analyzer isused to analyze one or more attributes of a waveform that is generatedin the sensor in response to each input frequency. In some embodiments,a frequency spectrum analyzer is used to analyze changes in a waveformthat is generated in the sensor as a function of time.

In some embodiments, the subject methods can be used to obtain resultsfrom a sample in a short period of time. For example, in someembodiments, the subject methods involve contacting a sensor with asample, and determining the concentration of an analyte in the sample ina period of time ranging from 5 seconds or less, up to 5 minutes, suchas 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 seconds, orup to 1, 1.5, 2, 2.5, 3, 3.5, 4, or 4.5 minutes.

Methods in accordance with embodiments of the invention include bothqualitative and quantitative analyte detection. As such, in someembodiments, the subject methods involve determining whether an analyteis present in a sample at a concentration that is above or below atarget, or threshold, concentration. In some embodiments, a thresholdconcentration for a particular analyte may range from 0.1 to 1,000 ppm,such as 0.5, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400,500, 600, 700, 800 or 900 ppm. In some embodiments, a thresholdconcentration for a particular analyte may range from 1 to 1,000 colonyforming units (CFU)/mL, such as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800 or 900 CFU/mL.

In some embodiments, the subject methods involve quantitativelydetermining the concentration of an analyte in a sample. In certainembodiments, the methods involving comparing a result obtained from asensor and/or an oscillator circuit to one or more calibration valuesthat can be used to quantitatively determine a concentration of ananalyte in a sample. In some embodiments, the subject methods involveapplying one or more calculations or algorithms to the results in orderto quantitatively determine a concentration of an analyte in a sample.In some embodiments, the subject methods involve comparing a pluralityof data obtained from a sensor and/or an oscillator circuit to ananalyte signature in order to determine whether the analyte is presentin the sample, and/or to quantitatively determine the concentration ofthe analyte in the sample.

In some embodiments, the subject methods involve verifying a resultobtained from a sensor by using a secondary detection device. Asdescribed above, in some embodiments, a sensor may include asurface-associated composition and/or a crosslinking composition thatincludes a detectable label or moiety. When an analyte crosslinks thesurface-associated compositions and crosslinking compositions on thesensor, the compositions are bound in place on the sensor. As such, incertain embodiments, the subject methods involve detecting and/orquantifying a detectable label or moiety on the sensor using a secondarydetection device. For example, in some embodiments, the subject methodsinvolve placing a used sensor in a secondary detection device, and usingthe secondary detection device to quantify the amount of a detectablelabel that is present on the sensor. Quantification of the detectablelabel on the sensor is used to verify the results that were previouslyobtained from the sensor.

In some embodiments, the subject methods involve monitoring the presenceof an analyte in a plurality of samples that are collected fromdifferent geographical locations. For example, in some embodiments, thesubject methods involve placing a plurality of sensors at differentgeographical locations, wherein each sensor is configured to contact asample at each geographical location. Each of the sensors analyzes asample from the geographical location where it is located, and theresults are transmitted to a detection unit for analysis. The resultsthat are obtained from each sensor can then be used to determine thepresence or absence of the analyte over a specific geographical area.

For example, in some embodiments, a plurality of sensors can be placedat different geographical locations in a body of water, such as a lake,stream or reservoir. Each sensor analyzes a sample from its geographiclocation and transmits the results to a detection unit for analysis. Theresults are then used to determine the presence or absence of theanalyte in various geographic portions of the body of water. Suchmethods find use is detecting, e.g., contamination of a body of waterwith pollutants or toxins, and more specifically, determining thespecific geographic portions of a body of water that are contaminatedwith a particular pollutant or toxin.

FIG. 2 depicts the same sensor as depicted in FIG. 1 shortly after asample containing an analyte 4 has been contacted with the sensor. Asshown in FIG. 2, the analyte 4 binds to the surface-associatedcompositions 2 as well as the crosslinking compositions 3.

FIG. 3 depicts the same sensor as depicted in FIG. 1 and FIG. 2 at alonger time after a sample containing an analyte 4 has been contactedwith the sensor. As shown in FIG. 3, the analyte 4 is bound to thesurface-associated compositions 2 as well as the crosslinkingcompositions 3, and the presence of the analyte 4 has resulted in theformation of crosslinks between the surface-associated compositions 2and the crosslinking compositions 3.

FIG. 4 depicts the same sensor as depicted in FIGS. 1-3 at an evenlonger time after a sample containing an analyte 6 has been contactedwith the sensor. As shown in FIG. 4, the analyte 4 is bound to thesurface-associated compositions 2 as well as the crosslinkingcompositions 3, and the presence of the analyte 4 has resulted in theformation of extensive crosslinks between the surface-associatedcompositions 2 and the crosslinking compositions 3.

Kits

Also provided are kits that at least include the subject systems anddevices or components thereof, e.g., as described above, andinstructions for how to use the devices in the detection andquantification of one or more target analytes in a sample. In someembodiments, a kit includes two or more sensors packaged in a sterilepackage. In some embodiments, the sensors in the kit are configured todetect different analytes.

In some embodiments, a kit includes one or more sensors that include adetectable label or moiety, and the kit includes a reagent that isconfigured to facilitate the detection of the detectable label with asecondary detection system. In some embodiments, a kit includes anactivated carbon composition. In certain embodiments, a kit includes anactivated carbon composition that includes a carbon nanotube.

In some embodiments, a kit includes one or more sample collectiondevices. Sample collection devices in accordance with embodiments of theinvention may include test tubes, cups, beakers, pipettes, dipsticks,swabs, spatulas, or other devices configured to collect at least a smallquantity of a sample. In some embodiments, the sample collection devicesmay include lids and/or caps for the devices, as well as suitablestorage containers, e.g., plastic storage bags or other packaging thatcan be used to store and/or transport the sample collection devicewithout contaminating the sample. Sample collection devices inaccordance with embodiments of the invention may be configured tocollect a liquid sample, a solid sample, or a semi-solid sample. In someembodiments, a sample collection device may be configured to collect asolid or semi-solid sample and to process the sample by crushing orpulverizing it to facilitate mixing the sample with a liquid.Accordingly, the subject sample collection devices may also includeimplements that are configured for mixing a solid or semi-solid samplewith a liquid.

The instructions for using the systems and devices as discussed aboveare generally recorded on a suitable recording medium. For example, theinstructions may be printed on a substrate, such as paper or plastic,etc. As such, the instructions may be present in the kits as a packageinsert, in the labeling of the container of the kit or componentsthereof (i.e. associated with the packaging or sub-packaging) etc. Inother embodiments, the instructions are present as an electronic storagedata file present on a suitable computer-readable storage medium, e.g.,a digital storage medium, e.g., a portable flash drive, a CD-ROM, adiskette, etc. The instructions may take any form, including completeinstructions for how to use the systems and devices, or as a websiteaddress with which instructions posted on the Internet may be accessed.

EXAMPLES Example 1: Detection of Viral Marker Protein using DeltaFrequency Measurement Only

AT-cut quartz crystals with a fundamental resonance frequency of 10 MHz(ICM, Oklahoma City, Okla.) were cleaned by soaking in 1M NaOH for onehour. The crystals were rinsed with distilled water and soaked for onehour in 1M HCl, then rinsed with distilled water, rinsed with 70%isopropyl alcohol, and allowed to air dry. After drying, 100 microlitersof a 1 mg/mL solution of recombinant protein G (BioVision, Inc.,Milpitas, Calif., Catalog No. 6512) were applied to the crystal surfacesand allowed to air dry. 100 microliters of a 0.010 mg/mL solution ofpolyclonal rabbit anti-ZEBOV VP40 antibody (Zaire strain of Ebola virus,IBT Bioservices, Gaithersburg, Md., Catalog No. 0301-010) were thenapplied on top of the dried protein G layer and allowed to air dry.

Application of the protein G and polyclonal antibody solutions to thecrystal created a plurality of surface-associated compositions that werestably associated with the surface of the quartz crystal, eachsurface-associated composition including a protein G molecule that wasadsorbed to the crystal surface, and a polyclonal antibody that wasbound to the immunoglobulin binding domain of the protein G molecule.Application of the protein G and polyclonal antibody solutions to thecrystal also created a plurality of crosslinking compositions, eachincluding one protein G molecule and two polyclonal antibodies, witheach polyclonal antibody bound to an immunoglobulin binding domain ofthe protein G molecule.

A sensor with the above-described compositions formed thereon wasoperatively connected to a system including a lever oscillator circuitand a frequency counter, and a baseline frequency of the sensor wasrecorded. The sensor was contacted with 100 microliters of salinesolution, and the change in frequency over time was recorded. A secondsensor was operatively connected to the system, and the baselinefrequency of the second sensor was recorded. The second sensor wascontacted with 100 microliters of a 0.01 mg/mL solution of recombinantZEBOV VP40 matrix protein (Zaire strain of Ebola virus, IBT Bioservices,Gaithersburg, Md., Catalog No. 0564-01), and the change in frequencyover time was recorded. These steps were repeated using a porcinegelatin solution (1 mg/mL) as a control instead of the recombinant ZEBOVVP40 matrix protein solution.

Both the saline solution and porcine gelatin solution controlsdemonstrated a mean change in frequency of 1.86±0.54 kHz (n=22) within 5seconds of applying the solution to the sensor. The sensor to which therZEBOV solution was applied demonstrated a mean change in frequency of9.98±0.47 kHz (n=10) within 5 seconds of applying the solution to thesensor. Based on the observed change in frequency of the sensor, therZEBOV solution was distinguished from the control solutions within 5seconds. As evidenced by the results from the porcine gelatin controlsolution, the sensor was specific for the rZEBOV protein, and did notgenerate a false positive signal when exposed to the porcine gelatinsolution.

Example 2: Detection of Viral Marker Protein using Delta FrequencyMeasurement Only

AT-cut quartz crystals with a fundamental resonance frequency of 10 MHz(ICM, Oklahoma City, Okla.) were cleaned by soaking in 1M NaOH for onehour. The crystals were rinsed with distilled water and soaked for onehour in 1M HCl, then rinsed with distilled water, rinsed with 70%isopropyl alcohol, and allowed to air dry. After drying, 100 microlitersof a 1 mg/mL solution of recombinant protein G (BioVision, Inc.,Milpitas, Calif., Catalog No. 6512) were applied to the crystal surfacesand allowed to air dry. 100 microliters of a 0.010 mg/mL solution ofpolyclonal rabbit anti-ZEBOV GP antibody (Zaire strain of Ebola virus,IBT Bioservices, Gaithersburg, Md., Catalog No. 0301-015) was thenapplied on top of the dried protein G layer and allowed to air dry.

Application of the protein G and polyclonal antibody solutions to thecrystal created a plurality of surface-associated compositions that werestably associated with the surface of the quartz crystal, eachsurface-associated composition including a protein G molecule that wasadsorbed to the crystal surface, and a polyclonal antibody that wasbound to the immunoglobulin binding domain of the protein G molecule.Application of the protein G and polyclonal antibody solutions to thecrystal also created a plurality of crosslinking compositions, eachincluding one protein G molecule and two polyclonal antibodies, witheach polyclonal antibody bound to an immunoglobulin binding domain ofthe protein G molecule.

A sensor with the above-described compositions formed thereon wasoperatively connected to a system including a lever oscillator circuitand a frequency counter, and a baseline frequency of the sensor wasrecorded. The sensor was contacted with 100 microliters of salinesolution, and the change in frequency over time was recorded. A secondsensor was operatively connected to the system, and the baselinefrequency of the second sensor was recorded. The second sensor wascontacted with 100 microliters of a 0.01 mg/mL solution and a 0.001mg/mL solution of recombinant EBOV rGPdTM protein (Zaire strain of Ebolavirus, IBT Bioservices, Gaithersburg, Md., Catalog No. 0501-015), andthe change in frequency over time was recorded. These steps wererepeated using a porcine gelatin solution (Sigma Aldrich, 1 mg/mL,Catalog No. G2500) as a control instead of the recombinant EBOV rGPdTMprotein solution.

Both the saline solution and porcine gelatin solution controlsdemonstrated a mean change in frequency of 1.86±0.54 kHz (n=22) within 5seconds of applying the solution to the sensor. The sensors to which the0.01 mg/mL solution of recombinant EBOV rGPdTM protein solution wasapplied demonstrated a mean change in frequency of 12.1±0.83 kHz (n=9).The sensors to which the 0.001 mg/mL solution of recombinant EBOV rGPdTMprotein solution was applied demonstrated a mean change in frequency of12.0±0.71 kHz (n=8). Based on the observed change in frequency of thesensor, the recombinant EBOV rGPdTM protein solutions were bothdistinguished from the control solution within 5 seconds. As evidencedby the results from the porcine gelatin control solution, the sensorswere specific for the recombinant EBOV rGPdTM protein, and did notgenerate a false positive signal when exposed to the porcine gelatinsolution.

Example 3: Determination of Delta Frequency Response from MonoclonalAntibody/Protein G Sensor as a Function of Salmonella HeidelbergConcentration

Sensors were made as described above by depositing 100 uL of a protein Gsolution (0.5 mg/mL, BioVision, Inc., Catalog No. 6510) on the surfaceof a piezoelectric base and allowing the solution to dry at 30° C. withcirculating air, then depositing 100 uL of anti-Salmonella Heidelberg(SH) murine monoclonal antibody solution (Cusabio, 10 ug/mL, Catalog No.CSB-PA472744YA01SWQ) on the piezoelectric base and allowing the solutionto dry at 30° C. with circulating air. A Salmonella Heidelberg controlsolution (UC Davis WIFSS, S. Heidelberg Strain #10) was diluted toprepare test solutions with Log CFU/mL values ranging from 0 to 9, asindicated in FIG. 10.

Each sensor was operatively connected to a system including a leveroscillator circuit and a frequency counter, and a baseline frequency ofthe sensor was recorded. The sensor was contacted with 100 microlitersof test solution, and the change in frequency over time was recorded.Four sensors were tested at each indicated concentration, and the dataare provided in FIGS. 9 and 10. A graph of delta frequency (Hz) v. Logconcentration was prepared for use as a calibration curve toquantitatively analyze a test sample with an unknown concentration ofSH. The correlation coefficient of the data was determined to be 0.9645,and the R² value was determined to be 0.9302.

Example 4: Analysis of Monoclonal Antibody/Protein G Sensor ReactionTime

Sensors were made as described above by depositing 100 uL of a protein Gsolution (0.5 mg/mL, BioVision, Inc., Catalog No. 6510) on the surfaceof a piezoelectric base and allowing the solution to dry at 30° C. withcirculating air, then depositing 100 uL of anti-Salmonella Heidelberg(SH) murine monoclonal antibody solution (Cusabio, 10 ug/mL, Catalog No.CSB-PA472744YA01SWQ) on the piezoelectric base and allowing the solutionto dry at 30° C. with circulating air. A Salmonella Heidelberg controlsolution (UC Davis WIFSS, S. Heidelberg Strain #10) was diluted toprepare test solutions with concentrations of 10³ CFUs/mL and 10⁷CFUs/mL.

Each sensor was operatively connected to a system including a leveroscillator circuit and a frequency counter, and a baseline frequency ofthe sensor was recorded. The sensor was contacted with 100 microlitersof test solution, and the change in frequency over time was recorded.Five sensors were tested at each indicated concentration and time point,and the data are provided in FIGS. 11 and 12. The data demonstrate thatthe signal from both concentration levels tested reached a plateau valueby the thirty second time point, and that a concentration of as low as10³ CFUs/mL could be detected in 30 seconds.

Example 5: Analysis of Polyclonal Antibody/Protein A Sensor ReactionTime

Sensors were made as described above by depositing 100 uL of a protein Asolution (1.0 mg/mL, BioVision, Catalog No. 6500B) on the surface of apiezoelectric base and allowing the solution to dry at 30° C. withcirculating air, then depositing 100 uL of anti-Salmonella rabbitpolyclonal antibody solution (AbD Serotec, 10 ug/mL or 100 ug/mL,Catalog No. 8209-4006) on the piezoelectric base and allowing thesolution to dry at 30° C. with circulating air. A Salmonella Heidelbergcontrol solution (UC Davis WIFSS, S. Heidelberg Strain #10) was dilutedto prepare a test solution with a concentration of 10³ CFUs/mL.

Each sensor was operatively connected to a system including a leveroscillator circuit and a frequency counter, and a baseline frequency ofthe sensor was recorded. The sensor was contacted with 100 microlitersof test solution or PBS, and the change in frequency was recorded afterthirty seconds. Three sensors were tested for each solution, and thedata are provided in FIG. 13. The data demonstrate that the SH testsolution (concentration of 10³ CFUs/mL) could be distinguished from thePBS solution based on delta frequency values at thirty seconds.

Example 6: Analysis of Contribution of Polyclonal Antibody/Protein GSensor Components to Signal Output

Sensors were prepared using different amounts of protein G solution (0.5mg/mL, BioVision, Inc., Catalog No. 6510) and anti-SalmonellaTyphimuriam (ST) polyclonal goat IgG antibody solution (KPL, Catalog No.01-91-99). Sensor #1 was prepared with polyclonal antibody only, and noprotein G. Sensor #2 was prepared with protein G only, and no polyclonalantibody. Sensor #3 was prepared with both protein G and polyclonalantibody. A Salmonella Typhimuriam control solution (KPL, Catalog No.50-74-01) with a concentration of 10⁵ CFUs/mL was used to test theresponse of each sensor.

Each sensor was operatively connected to a system including a leveroscillator circuit and a frequency counter, and a baseline frequency ofthe sensor was recorded. The sensor was contacted with 100 microlitersof test solution, and the change in frequency over time was recorded.Three sensors were tested at each indicated concentration and timepoint, and the data are provided in FIG. 14. The data demonstrate thatsensor #3, which included both protein G and anti-SH polyclonalantibody, provided the strongest delta frequency response, and that thisresponse reached a plateau value by approximately 30 seconds.

FIG. 15 provides data showing the delta frequency response value atthirty seconds after depositing 100 uL of 10⁵ CFUs/mL test solution onsensors having the indicated amounts of anti-ST polyclonal antibody andprotein G or protein A. Three sensors were evaluated for each componentcombination. The highest delta frequency value (25,828 Hz) was obtainedfrom a sensor that was made from a 100 ug/mL anti-ST polyclonal antibodysolution and a 3 mg/mL protein G solution.

As provided in FIG. 15, one sensor type was prepared using protein A(instead of protein G) and the anti-ST polyclonal goat IgG antibody.Protein A does not bind to goat IgG antibodies, and as such, sensorswith this combination of components served as a negative control. Thedata demonstrate that the sensors made with protein A did not provide astrong a delta frequency signal when contacted with the test solution,as was expected.

Example 7: Sensor Utilizing Biotin/Streptavidin Components

Sensors were prepared using a biotin-protein G solution (100 ug/mL or 10ug/mL, BioVision, Inc., Catalog No. 6512) whose protein G molecules arebiotinylated with approximately 2 biotin molecules per protein Gmolecule. 100 uL of the biotin-protein G solution was deposited on thesurface of a piezoelectric base and allowed to dry at 30° C. withcirculating air to form the surface-associated compositions. Next, 100uL of the same biotin-protein G solution was deposited on top of thesurface-associated compositions and allowed to dry at 30° C. withcirculating air to form the crosslinking compositions. A schematicillustration of biotin-protein G sensor is provided in FIG. 16. In thedepicted embodiment, the piezoelectric base 1 is shown, as well as aplurality of surface-associated compositions 2 and a plurality ofcrosslinking compositions 3. Each surface-associated composition 2includes a protein G molecule and two or more biotin molecules. Eachcrosslinking composition 3 also includes a protein G molecule and two ormore biotin molecules. A streptavidin control solution (streptavidin inPBS, Jackson Immunoresearch, Catalog No. 016-000-113) was used as a testsolution. Each molecule of streptavidin can bind to four molecules ofbiotin, thereby crosslinking the surface-associated compositions and thecrosslinking compositions.

Each sensor was operatively connected to a system including a leveroscillator circuit and a frequency counter, and a baseline frequency ofthe sensor was recorded. 100 uL of a 1 mg/mL or 0.1 mg/mL streptavidintest solution, or a phosphate buffered saline (PBS) control solution,were applied to each sensor, and the delta frequency value was measuredat 30 seconds. Three sensors were tested with PBS, and four sensors weretested with each streptavidin solution, as indicated in FIGS. 17 and 18.The data demonstrate that the PBS solution could be distinguished fromthe streptavidin solutions after thirty seconds. Sensors that were madeusing the 100 ug/mL biotin-protein G solution (as opposed to the 10ug/mL biotin-protein G solution) showed a greater change in deltafrequency when contacted with streptavidin solution v. PBS.

Example 8: Polyclonal Antibody/Protein G Sensor Delta Frequency Responsein Liquid State and Dry State

Liquid state sensors, in which the components on the surface of thesensor are hydrated, rather than dried, were prepared by depositing 100uL of a 1 mg/mL protein G solution (BioVision, Inc., Milpitas, Calif.,Catalog No. 6512) onto the surface of a piezoelectric base andincubating for 22° C. for 30 minutes. Next, 100 uL of a 100 ug/mLanti-Salmonella Typhimuriam (ST) polyclonal antibody solution (KPLLaboratories, Catalog No. 01-91-99) was deposited on the surface of thepiezoelectric base, and the sensor was again incubated at 22° C. for 30minutes. The delta frequency of the liquid state sensor was measured andrecorded.

Dry sensors were prepared as described above by applying the protein Gsolution on the surface of a piezoelectric base and allowing thesolution to dry at 30° C. with circulating air, then depositing 100 uLof anti-Salmonella Heidelberg (SH) polyclonal antibody solution on thepiezoelectric base and allowing the solution to dry at 30° C. withcirculating air.

Each sensor was operatively connected to a system including a leveroscillator circuit and a frequency counter, and a baseline frequency ofthe sensor was recorded. 100 uL of a Salmonella Typhimuriam (ST) controlsolution (KPL, Catalog No. 50-74-01) with a concentration of 10⁵CFUs/mL, or 100 uL of phosphate buffered saline (PBS), was used to testthe response of each sensor. To correct for any mass effects experiencedby the liquid state sensor, 300 uL of the ST control solution, or 300 uLof PBS, was also used to test the sensor response. Three sensors weretested for each solution (n=3). The data are provided in FIG. 19. Thedata indicate that the dry sensors provide a larger delta frequencyvalue as compared to the liquid state sensors.

Example 9: Determination of Limit of Detection (LOD) of Salmonella viaELISA

The limit of detection (LOD) was measured for two different Salmonellaserovars using a standard ELISA (enzyme linked immune-sorbent assay).The first was Salmonella Enteritidis (SE) and the second was SalmonellaHeidelberg (SH). The ELISA was conducted using conjugated biotinylatedantibodies with horse radish peroxidase (HRP).

Field strains of SE and SH were grown independently in Tryptic Soy Broth(TSB; Difco, BD) at 37° C. with orbital rotation at 50 rpm for 5 hfollowed by a refrigeration step at 6° C. for 2 h to stabilize thebacterial growth. Bacterial cells were removed from the growth media(washed) with centrifugation at 10,000 rpm for 10 min; the supernatant(TSB) was discarded and the cells were re-suspended in PhosphateBuffered Saline (PBS; Sigma-Aldrich, St. Louis, Mo.). Cell counts wereestimated using a regression equation that extrapolated bacterialconcentration from the optical density at 600 nm; 1 mL of the washedcell-suspension was 10-fold serial diluted in PBS to obtain the targetconcentrations of 10⁶-10² CFU/mL. Cell counts were confirmed by spreadplating the serial dilutions onto tryptic soy agar (TSA; Difco, BD) andincubating at 37° C. for 18 to 24 h.

Both capture antibodies (SH was a polyclonal and SE was a monoclonal)were diluted in 0.2 M sodium carbonate/bicarbonate, pH 9.4. Then 100 uLof capture antibody were added to each well. The plate was covered andincubated at 4° C. The next day, the solution was removed and washedwith 200 uL of PBS in each well, three times for 5 minutes each on ashaking platform. After washing, 300 uL of blocking buffer (2% BSA inPBS) were added to each well and incubated overnight at 4° C. The nextday, the blocker was removed and 100 uL of antigen were added to eachwell. The plate was then incubated at room temperature for 1 hour. Thesamples were removed and the plate was washed with PBS. Afterwards, 100uL of the biotinylated detection antibody was added to each well and theplate was incubated at room temperature for another hour. Then thesolution was removed and the plate was washed with PBS three times.Next, streptavidin-HRP served as the enzyme conjugate and 100 uL wereadded to each well. The plate was then covered and incubated at roomtemperature for 1 hour. The solution was removed and the plate waswashed 6 times with PBS. TMB served as the substrate solution and 100 uLwere added to each well and the plate was incubated at room temperature.After 15 minutes, the absorbance at 650 nm was taken using aspectrometer.

The results established that the LOD for SE was 10⁴, and the LOD for SHwas 10⁶ CFUs/mL when measured via ELISA. The SE and SH captureantibodies cross reacted against the SH and SE antigens at 10⁷ CFUs/mL.However, the OD₆₅₀ values were lower for the cross-reacted antigen thanfor the target antigen. The results are provided in FIG. 20. The topnumber in each cell is the OD₆₅₀ value without subtracting the blankvalue, and the bottom number is the OD₆₅₀ value with the blank valuesubtracted.

Example 10: Use of Activated Carbon to Increase Detection

Sensors were made as described above by applying the protein G solutionon the surface of a piezoelectric base and allowing the solution to dryat 30° C. with circulating air, then depositing 100 uL ofanti-Salmonella Typhimuriam antibody solution (10 ug/mL, KPL Labs,Catalog No. 01-91-99) on the piezoelectric base and allowing thesolution to dry at 30° C. with circulating air. In certain sensors,approximately 1 ug of activated, micronized carbon (Cabot Corp.) wasapplied to and evenly dispersed over the piezoelectric base duringproduction. A Salmonella Typhimuriam positive control (ST pos. control)with a concentration of 10⁵ CFUs/mL (KPL Labs, Catalog No. 50-74-01),phosphate buffered saline (PBS) solution, or activated carbon solutionwas used to test the response of the sensors.

Each sensor was operatively connected to a system including a leveroscillator circuit and a frequency counter, and a baseline frequency ofthe sensor was recorded. The sensor was contacted with 100 microlitersof test solution (either ST pos. control, PBS, or activated carbonsolution), and the change in frequency over time was recorded. The dataare provided in FIG. 21. The results show that the inclusion ofactivated carbon in the sensor increases the mean delta frequency ofresponse by approximately 5×.

Example 11: Method for Determining Analyte Concentration in an UnknownSample using an Internet-enabled System

FIG. 22 is flow diagram illustrating the steps involved with using anInternet-enabled system to determine the concentration of a targetanalyte in an unknown sample. First, a sensor is connected to adetection component. Sensor identification information stored on acomputer readable medium on the sensor is read by the detectioncomponent and communicated to the computer. The computer thencommunicates with an operations database via the Internet to acquirecalibration information and test algorithm information for theparticular sensor. Next, the sensor is contacted with a samplecontaining an unknown concentration of a target analyte. The detectioncomponent applies the test algorithm and the raw data is analyzed. Thecomputer applies the calibration information to the raw data todetermine the concentration of the target analyte in the sample. Theresults are communicated to a user via a GUI on the detection component.The results are also transmitted to a results database via the Internet,where the results are archived.

Example 12: Method for Determining Analyte Concentration in an UnknownSample using a Non-Internet-enabled System

FIG. 23 is flow diagram illustrating the steps involved with using anon-Internet-enabled system to determine the concentration of a targetanalyte in an unknown sample. First, a sensor is connected to adetection component. Sensor identification information stored on acomputer readable medium on the sensor is read by the detectioncomponent. The sensor identification information includes calibrationinformation as well as test algorithm information that is used by thedetection component when analyzing a sample. Next, the sensor iscontacted with a sample containing an unknown concentration of a targetanalyte. The detection component applies the test algorithm and the rawdata is analyzed. The detection component applies the calibrationinformation to the raw data to determine the concentration of the targetanalyte in the sample. The results are communicated to a user via a GUIon the detection component.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

1. A sensor comprising: a piezoelectric base; a plurality ofsurface-associated compositions that are stably associated with thepiezoelectric base; and a plurality of crosslinking compositions thatare configured to crosslink one or more surface-associated compositionsin the presence of an analyte.
 2. The sensor according to claim 1,wherein the piezoelectric base comprises at least one electrode.
 3. Thesensor according to claim 1, further comprising an oscillator circuitthat is electrically connected to the at least one electrode, and thatis configured to drive the sensor at one or more frequencies.
 4. Thesensor according to claim 1, wherein the oscillator circuit comprises anautomatic gain control (AGC) portion.
 5. The sensor according to claim1, wherein the surface-associated compositions comprise one or more of:protein A, protein G, protein A/G, or protein L.
 6. The sensor accordingto claim 1, wherein the surface-associated compositions comprise one ormore polyclonal antibodies.
 7. The sensor according to claim 1, whereinthe crosslinking compositions comprise one or more of: protein A,protein G, protein A/G, or protein L.
 8. The sensor according to claim1, wherein the crosslinking compositions comprise one or more polyclonalantibodies.
 9. The sensor according to claim 1, further comprising acomputer-readable medium that contains a plurality of stored data. 10.The sensor according to claim 1, wherein the stored data comprises acalibration value for the sensor.
 11. The sensor according to claim 1,wherein the stored data comprises an analyte signature.
 12. The sensoraccording to claim 1, wherein the stored data comprises an operatingparameter for the sensor.
 13. The sensor according to claim 1, whereinthe piezoelectric base comprises a quartz crystal.
 14. The sensoraccording to claim 1, wherein the quartz crystal is an AT-cut quartzcrystal.
 15. The sensor according to claim 1, wherein the piezoelectricbase comprises a surface texture.
 16. The sensor according to claim 1,wherein the at least one electrode comprises an interdigitatedstructure.
 17. The sensor according to claim 1, wherein a plurality ofthe surface-associated compositions and/or crosslinking compositionscomprises a detectable label.
 18. A system for detecting the presence ofan analyte in a sample, the system comprising: a sensor comprising: apiezoelectric base; a plurality of surface-associated compositions thatare stably associated with the piezoelectric base; a plurality ofcrosslinking compositions that are configured to crosslink one or moresurface-associated compositions in the presence of the analyte; and atleast one electrode; an oscillator circuit that is electricallyconnected to the at least one electrode and is configured to drive thesensor at one or more frequencies, wherein the oscillator circuitcomprises an automatic gain control (AGC) portion; a detection unitconfigured to receive a plurality of data from the oscillator circuit;and a processor configured to analyze the data received from theoscillator circuit and to detect the presence of the analyte in thesample. 19.-44. (canceled)
 45. A method for detecting the presence of ananalyte in a sample, the method comprising: contacting a sensor with thesample, wherein the sensor comprises: a piezoelectric base; a pluralityof surface-associated compositions that are stably associated with thepiezoelectric base; a plurality of crosslinking compositions that areconfigured to crosslink one or more surface-associated compositions inthe presence of the analyte; and at least one electrode; applying acurrent to an oscillator circuit that is electrically connected to theat least one electrode and is configured to drive the sensor at one ormore frequencies, wherein the oscillator circuit comprises an automaticgain control (AGC) portion; measuring one or more parameters of thesensor and/or oscillator circuit as a function of time; and analyzingthe one or more parameters of the sensor and/or oscillator circuit todetect the presence of the analyte in the sample. 46.-67. (canceled) 68.A kit comprising two or more sensors according to claim 1, wherein thetwo or more sensors are packaged in a sterile package. 69.-73.(canceled)